Maii: 


(7 

LIBRARY 


UNIVERSITY  OF  CALIFORNIA. 


Deceived 


,  189(0 


,**>-,  BIOL06Y 

Accession  No.  £>  J  3J  / .  Class  No.     LIBRARY. 


ELEMENTARY  PRACTICAL 
BACTERIOLOGY 


A  COUKSE 

OP 

ELEMENTARY  PRACTICAL 
BACTEBIOLOGY 

INCLUDING 

BACTEKIOLOGICAL  ANALYSIS  AND 
CHEMISTKY 


BY 

A.  A  KANTHACK,  M.D.,  M.E.C.P. 

LECTURER  ON  PATHOLOGY  AND  BACTERIOLOGY,  AND  CURATOR  OF  THE  MUSEUM, 
ST.  BARTHOLOMEW'S  HOSPITAL 

AND 

J.  H.  DBYSDALE,  M.B.,  M.K.C.P. 

CASUALTY   PHYSICIAN,   ST.   BARTHOLOMEW'S  HOSPITAL;  LATE  DEMONSTRATOR  OF 
BACTERIOLOGY,    UNIVERSITY   COLLEGE.   LIVERPOOL 


iLontron 

MACMILLAN    AND    CO. 

AND  NEW  YORK 
1896 

All  rights  reserved 


Main  Lib. 


\< 


BIOLO«Y 

LIBRARY 

6 


First  Edition  February  1895 
Reprinted  with  corrections  1895,  1896 


PEEFACE 

BACTERIOLOGY  is  an  essential  branch  of  Pathology,  of  such 
importance  that  it  is  gradually  becoming  a  necessary,  if 
not  a  compulsory,  element  in  the  study  of  Medicine.  As 
a  matter  of  fact,  most  Faculties  and  Universities  granting 
a  Diploma  or  Degree  in  Public  Health  demand  three 
months'  practical  instruction  in  Bacteriology.  It  is  only 
by  patient  laboratory  work  that  Bacteriology  can  be 
taught  in  such  a  manner  as  to  serve  any  useful  purpose. 

One  of  the  chief  difficulties  in  the  practical  instruction 
which  has  presented  itself  to  us  has  been  the  want  of  a 
suitable  handbook  for  the  laboratory.  The  student  has 
felt  this  want  as  much  as  his  teachers.  He  cannot  be 
expected  to  dig  the  necessary  information  out  of  large 
and  voluminous  text-books,  which,  moreover,  are  often 
deficient  in  all  practical  details.  At  St.  Bartholomew's 
Hospital  we  have  been  in  the  habit  of  giving  out  slips  with 
full  directions  about  the  work  to  be  done  from  day  to  day. 
This  method  entails  a  certain  amount  of  disagreeable 


VI  PREFACE 


trouble  on  the  part  of  the  lecturer,  and  finds  only  partial 
favour  with  the  student. 

We  have  therefore  ventured  to  collect  and  put  together 
the  slips  and  notes  in  the  form  and  shape  of  a  small  hand- 
book, which,  though  originally  intended  for  use  at  the 
laboratories  of  St.  Bartholomew's  Hospital,  we  hope  may 
supply  a  want  felt  elsewhere.  The  work  is  divided  into 
Lessons,  and  the  book  is  arranged  in  three  parts,  of  which 
Parts  I.  and  II.  (Elementary  Bacteriology  and  Bacteriologi- 
cal Analysis)  encompass  three  months'  work,  as  required 
for  the  Diploma  of  Public  Health  at  London  and  Cambridge. 
Naturally,  we  have  adhered  to  our  own  system  of  teaching, 
which  represents  that  used  for  some  years  at  the  Pathologi- 
cal Laboratories  of  Cambridge  University  and  St.  Bartholo- 
mew's Hospital.  A  few  explanatory  remarks,  therefore, 
appear  necessary. 

At  St.  Bartholomew's  Hospital  we  invariably  begin  with 
an  Elementary  Course  (Part  I.),  which  extends  over  five  to 
six  weeks  (three  lessons  a  week).  All  media  are  supplied, 
so  that  the  simpler  methods  of  work,  inoculations,  cultiva- 
tions, staining,  etc.,  may  be  quickly  acquired  and  the 
student  may  become  acquainted  with  the  general  principles. 
The  rest  of  the  term  is  then  devoted  to  Bacteriological 
Analysis  (Part  II),  which  includes  the  preparation  of  the 
various  media,  and  the  analysis  of  air,  water,  food,  etc., 
the  examination  of  filters,  disinfectants,  etc.  As  far  as 
possible  we  have  given  full  directions,  but  these  Lessons 
are  not  intended  to  supplant  the  demonstrator,  nor  do 


PREFACE  vii 


they  pretend  to  cover  the  whole  ground  of  Practical 
Bacteriology.  In  three  months  it  is  impossible  to  teach 
or  to  learn  much.  Hence  we  have  attempted  to  make  the 
Lessons  as  representative  as  possible,  and  to  introduce  the 
student  to  the  more  important  analytical  studies  and  prob- 
lems. Parts  I.  and  II.  are  especially  arranged  to  suit  the 
requirements  of  candidates  for  one  or  other  Diploma  in 
Public  Health. 

We  have  always  described  methods  which  we  use  our- 
selves, and  in  all  cases  we  have  given  frankly  and  without 
reserve  what  we  consider  to  be  the  quickest  or  the  best 
method.  Every  laboratory  has  its  own  ways  and  means, 
its  "short  cuts,"  and  we  might  almost  say  its  "secrets." 
We  have  divulged  all  our  own,  not  because  we  flatter  our- 
selves that  there  are  no  better  methods,  but  because  we  have 
found  from  practical  experience  that  most  methods  depend 
in  their  execution  on  some  small  detail  which  is  frequently 
withheld  from  the  beginner.  These  so-called  "  laboratory 
tips,"  which  are  acquired  through  experience  and  practice, 
are  often  omitted  in  descriptions  of  special  methods, 
because  they  seem  unimportant,  or  possibly  too  precious. 
Methods  which  require  acquaintance  with  glass-blowing, 
or  which  recommend  themselves  readily  enough  to  old 
hands  but  seem  unsuited  to  the  inexperienced,  have  been 
purposely  left  out. 

We  confess  at  once  that  the  methods  described  in  these 
Lessons  for  the  most  part  are  not  our  own.  They  have 
been  carried  away  from  the  various  laboratories  in  which 


vni  PREFACE 


it  has  been  our  privilege  to  work.  Often,  no  doubt,  they 
have  been  somewhat  modified  or  rearranged  for  the  purpose 
of  instruction.  We  feel,  however,  that  we  must  give  ex- 
pression to  our  indebtedness  to  Dr.  E.  Klein  for  many  hints 
and  methods  which  were  legitimately  acquired  by  us  from 
his  laboratory.  Many,  if  not  most,  of  the  useful  methods 
of  Bacteriological  Analysis  current  in  this  country  have 
emanated  thence,  and  we  therefore  do  not  hesitate  to 
acknowledge  gratefully  all  the  assistance  which  consciously 
or  unconsciously  we  have  derived  from  that  liberal  source. 
The  third  part  of  this  little  book  forms,  so  to  speak,  an 
appendix  to  the  two  previous  parts,  and  comprises  an  intro- 
duction to  Bacteriological  Chemistry  for  those  who  are 
desirous  of  devoting  some  of  their  time  to  advanced  in- 
vestigation and  research  work.  Bacteriological  Chemistry 
is  yet  a  young  and  unfledged  science,  even  more  unsatis- 
factory than  Physiological  Chemistry.  We  have  therefore 
selected  a  few  points  in  the  shape  of  exercises  which  are 
either  of  classical  importance  or  essential  for  research. 

A.  A.  K. 
J.  H.  D. 

LONDON,  Zlst  December  1894. 


CONTENTS 

PAET    I 
GENERAL  BACTERIOLOGY 

LESSON  I 

PAGE 

METHODS  OF  INOCULATION  — 

(1)  Agar-Agar  Streak  Cultures  ......  3 

(2)  Action  of  Sunlight  on  the  Bacillus  pyocyaneus     .         .  4 

(3)  Action  of  Sunlight  on  the  Bacillus  prodigiosus     .         .  4 

(4)  Action  of  Oxygen  on  the  Bacillus  prodigiosus       .         .  5 

(5)  Potato  Cultures  ........  6 

(6)  Agar-Agar  Cultures  of  the  Bacillus  fluorescens      .        .  6 

(7)  Gelatine  Streak  Cultures      ......  6 


(9) 


|  Gelatine  Stab  Cultures       ......     6,7 


LESSON    II 


TYPES  OF  MICRO-ORGANISMS 8 

STAINING  OF  MICRO-ORGANISMS  WITH  SIMPLE  BASIC  ANILINE 
DYES— 

(1)  Simple  Aniline  Dyes             9 

(2)  Preparation  of  Cover-glass  Films 9 

(3)  Staining  of  Films 10 


CONTENTS 


LESSON   III 

PAGE 

STAINING  OF  MICRO-ORGANISMS  (continued) — 

Loffler's  Methylene-Blue 12,  13 

EXAMINATION  OF  MOULDS — 

Aspergillus  niger 13,  14 

HANGING  DROP  CULTURES— 

(a)  Streptococcus  pyogenes        .         .         .         .         .         .         14 

(6)  Bacillus  filamentosus 14 

EXAMINATION  OF  BACTERIA  IN  A  HANGING  DROP — 

Motility  of  Micro-organisms 15,  16 

LESSON   IV 

STAINING  OP  HANGING  DROP  CULTURES — 

(a)  Loffler's  Methylene-Blue 17 

(6)  Gram's  Method 17-19 

GRAM'S  METHOD 17-19 

STAINING  OF  Pus — 

(a)  Loffler's  Methylene-Blue 20 

(&)  Gram's  Method 20 

INOCULATIONS  WITH  THE  BACILLUS  ANTHRACIS— 

Potato  ;  Broth  ;  Agar-Agar  ;  Gelatine       ....  21 

CURDLING  FERMENT 21 

LESSON   V 

BACILLUS  ANTHRACIS  (continued) — 

Mode  of  Growth  on  Various  Media 22 

Hanging  Drop  Cultures 22 

ASPOROGENOUS  CULTURES— 

(a)  Carbolic  Acid  Broth 22 

(6)  Heat 23 


CONTENTS  xi 


PAGE 

ACID  FORMATION  BY  VIRULENT  AND  ATTENUATED  BACILLI.  23 

METHODS  OF  ATTENUATION     .......  23 

STAINING  OF  THE  BACILLUS  ANTHRACIS — 

(a)  Loffler's  Methylene-Blue  23 

(6)  Gentian-Violet 23 

(c)  Gram's  Method 24 


LESSON   VI 

BACILLUS  ANTHRACIS  (continued) — 

Hanging  Drop  Cultures  (Staining)  „        25 

Asporogenous  Cultures  (Staining) 25 

Impression  Specimens .26 

Examination  of  Fresh  Tissues  by  Means  of  Films — 
Spleen  of  Mouse 27,  28 

LESSON   VII 

BACILLUS  ANTHRACIS  (continued)— 

Plate  Culture  Method         .         .         .         .        „  '      .         .  29,  30 
Staining  of  Spores      .  30-32 

Ehrlich's,  Loffler's,  or  Ziehl's  Fuchsine      ....    30-32 

SEPARATION  OF  HAY  BACILLUS  OUT  OF  HAY  INFUSION  33 


LESSON   VIII 

BACILLUS  ANTHRACIS  (concluded) — 

Impression  Specimens  (Gelatine  Plates)     ....         34 

STAINING  OF  TISSUES  IN  FROZEN  SECTIONS — 

(a)  Loffler's  Methylene-Blue 35 

(&)  Methylene-Blue  and  Eosine 35,  36 

(c)  Methylene-Blue  and  Picrocarmine          ...         36 

(d)  Gram's  Method     .         -         -         ,         .         .         .  36,  37 


xii  CONTENTS 


CHOLERA — 

Inoculation    of    Gelatine,   Agar-Agar,    and    Broth    with 

Various  Vibrios 37,  38 

STAINING  OF  FLAGELLA  (VAN  ERMENGEM)— 

(a)  Bacillus  of  Typhoid  Fever  .        .        .        .        .        .  38,  39 

(b)  Vibrio  cholera  Asiatics? 38,  39 


LESSON   IX 

CHOLERA  (continued) — 

Examination   of  Vibriones   choleras    Asiaticse,   Finkler- Prior, 

and  Metchnicovi  in  Cultures 40 

STAINING  OF  VIBRIOS — 

(a)  Gentian-Violet 40 

(Z>)  Fuchsine 40 

SPIRILLUM  RUBRUM — 

(a)  Staining 41 

(b)  Gelatine  Stab  Cultures 41 

Hanging  Drop  Cultures  of  Vibrio  choleras  Asiaticse           .         .  41 

CHOLERA  VIBRIOS  IN  THE  ANIMAL  BODY — 

(a)  Peritoneal  Fluid  of  Guinea-Pig 41 

(b)  Separation  of  Vibrios  by  Plate  Cultures        .         .         .42,  43 
ACTION  OF  SUNLIGHT  ON  THE  VIBRIO  CHOLERA  ASIATICS  .        43 


LESSON   X 

CHOLERA  (concluded) — 

Examination  of  Plates 44 

Impression  Specimens 44 

Varieties  of  Choleraic  Vibrios 44,  45 


CONTENTS  xiii 


ACTINOMYCOSIS — 

Cladothrix  nivea 45 

Celloidin  Sections  of  Actinomycosis   .         .         .         .         .  45,  46 

Mycetoma 46 

FLAGELLA  STAINING  (AFTER  PITFIELD)  46 

LESSON   XI 

PYOGENIC  Cocci — 

Cultures  in  Broth,  Agar-Agar,  and  Gelatine       .  .  47,  48 

Hanging  Drop  Cultures 48 

Staining  of  Cultures 48 

Staining  of  Pus 48.  49 

Staining  of  Gonorrhoeal  Pus       •        .         •         -         .         .         49 

LESSON   XII 

PYOGENIC  Cocci  (concluded} — 

Staining  of  Hanging  Drops        .                                   e  .         50 
Staining  in  Frozen  Sections — 

(a)  Pyaemia        .                  .51 

(b)  Erysipelas .51 

(c)  Pneumonia ,  .        51 

(d)  Micrococcus  tetragonus 51 

FIBRIN  STAINING  ("WEIGERT'S  METHOD)        .        .        .  .  51,  52 


LESSON   XIII 

TYPHOID  FEVER— 

Cultures  of  the  Bacillus  of  Typhoid  Fever.         .         .        .53,54 

Cultures  of  the  Bacterium  coli  commune    .         .         ,         .  53,  54 
Shake  Cultures          ........         54 

Varieties  of  the  Bacterium  coli  commune  .         .         .         .  54,  55 

Typhoid  Spleen  (Frozen  Sections) 55 


xiv  CONTENTS 


PAGE 

PNEUMOCOCCUS  :  STAINING  OF  CAPSULE         ....        55 
Staining  of  Carbolised  Pneumonic  Sputum         ...         56 

DIPHTHERIA — 

Cultures  of  Diphtheria  Bacillus          .         .         .        .         .  56,  57 

Staining  of  Cover-glass  Films 57 

Diphtheritic  Membrane 57 

Hanging  Drop  Cultures 57 

LEPROSY — 

Staining  of  Frozen  Sections       .        .        .  '  ,         .  57,  58 


LESSON   XIV 

TUBERCULOSIS — 

Staining  of  Fresh  Tubercular  Sputum        ....   59-61 
Staining  of  Carbolised  Sputum 61,  62 

STAINING  OF  FROZEN  SECTIONS  OF  TUBERCULOUS  TISSUES — 

(a)  Quick  Method 62,  63 

(6)  Slow  Method 63,  64 

STAINING  OF  PARAFFIN  SECTIONS  OF  TUBERCULOUS  TISSUES — 

(a)  Slow  Method 64,  65 

(b)  Quick  Method 65 

STAINING  OF  PARAFFIN  SECTIONS  OF  DIPHTHERITIC  MEMBRANE — 

(a)  Eosine  Methylene-Blue 65 

(b)  Weigert's  Fibrin  Method 66 


LESSON   XV 

TUBERCULOSIS  (concluded) — 

(a)  Mammalian  Tubercle  (Pure  Cultures)  67 

(b)  Avian  Tubercle  (Pure  Cultures) 67 


CONTEXTS  xv 


GLANDERS — HORSE'S  LUNG  (PARAFFIN  SECTIONS) — 

(a)  Slow  Method 68 

(6)  Quick  Method .         .  68,  69 

TETANUS  BACILLI 69 

ACTINOMYCOSIS  (PARAFFIN  SECTIONS)— 

(a)  Weigert's  Fibrin  Method 69 

(b)  Differential  Staining  of  Clubs       .....         70 
PY^MIC  SPLEEN  (PARAFFIN  SECTIONS) — 

Gram's  Method 70,  71 

PHAGOCYTOSIS— 

(a)  Hanging  Drop 71,  72 

(b)  Stained  Specimens 72,  73 

(c)  Local  Immunity  and  General  Infection         .         .         .  73,  74 

(d)  Effect  of  Heat  on  Phagocytosis 74 

LESSON    XVI 

PHAGOCYTOSIS  (concluded} — 

(a)  Heated  Frog 75 

(6)  Anaesthetised  Frog 76 

(c)  Phagocytosis  in  a  Hanging  Drop  .         .         .         .  76,  77 

CHEMIOTAXIS 77 

DIAGNOSIS  OF  DIPHTHERIA 78 


CONTENTS 


PAET  II 
BACTEEIOLOGICAL  ANALYSIS 

LESSONS  I  AND  II 

PAGE 

CLEANING  AND  STERILISATION  OF  GLASS — 

(1)  Cleaning  of  New  Test-Tubes 81 

(2)  Cleaning  of  Used  Test-Tubes 82 

(3)  Cleaning  of  Flasks  and  Beakers 82,  83 

(4)  Sterilisation 83 

PREPARATION  OF  NUTRIENT  MEDIA — 

(1)  Beef  Broth 83-85 

(2)  Glycerine  Broth 85 

(3)  Grape-Sugar  Broth      ...„=..         85 

(4)  Meat  Infusion      ........         86 

(5)  Gelatine •  .  86,  87 

(6)  Grape-Sugar  Gelatine  .......         87 

(7)  25  Per  Cent  Gelatine 87,  88 

(8)  Carbolic  Acid  Gelatine          .         .         .         .         .         .         88 

(9)  Peptone  Solution         .        .        .        .        .        .        .  88,  89 

(10)  Potato  Tubes 89 

(11)  Milk  Tubes 89 

(12)  Agar-Agar 90,  91 

(13)  Glycerine  Agar-Agar .         91 

(14)  Grape-Sugar  Agar-Agar 91,  92 

(15)  Blood  Serum  Tubes  92 


CONTENTS  xvii 


LESSON    III 

PAGE 

EXAMINATION  OF  WATER — 

I.  Quantitative  Examination. 

A.  Tap  Water— 

(a)  Plate  Culture  Method   ......    93-95 

(6)  Roll  Tubes .        .         .  95,  96 

B.  Distilled  Water 96 

C.  Tank  Water 96 

D.  Effect  of  Sunlight  on  Water 97 

LESSON    IV 

EXAMINATION  OF  WATER  (continued) — 

II.  Qualitative  Examination. 

A.  Bacillus  of  Enteric  Fever  and  Bacterium  coli  commune   98-100 

LESSON    V 

EXAMINATION  OF  WATER  (concluded) — 
//.   Qualitative  Examination  (concluded). 

B.  Vibrio  cholerse  Asiatics?  — 

I.   Peptone  Method 101-103 

II.  Gruber's  Method 103 

III.  Agar-Agar  Plates  .  ...  103,  104 

IV.  Gelatine  Plates 104 

LESSON   VI 

EXAMINATION  OF  MILK — 

/.  Quantitative  Examination      .         .         ,         .         ,         .          105 
//.   Qualitative  Examination — 

(1)  Bacillus  of  Enteric  Fever  .         .  ...          106 


xvni  CONTENTS 


PAGE 

(2)  Bacterium  coli  commune 106 

(3)  Streptococcus  pyogenes     .         .         .         .         .         .          106 

(4)  Bacillus  diphtherias 106 

(5)  Bacillus  tuberculosis  (Van  Ketel's  Method)        .         .  107, 108 


LESSON    VII 

EXAMINATION  OF  Am  AND  DUST — 

I.  Plate  Culture  Method 109 

II.  Aspiration  through  Broth       .         .         .         .         .          110 
III.  Filtration  through  Sugar 110,  111 


LESSON    VIII 

EXAMINATION  OF  AIR  AND  DUST  (concluded] — 

IV.  Anaerobic  Germs  in  Air  and  Dust    ....  113-115 
EXAMINATION  OF  SOIL — 

A.  Surface  Soil— 

(a)  Aerobic  Germs 115 

(b)  Anaerobic  Germs 115,  116 

B.  Tetanus  and  Malignant  (Edema  Bacilli — 

(a)  Growth  in  an  Exhausted  Flask          .         .         .         .          116 

(b)  Growth  in  Hydrogen 116-118 

(c)  Fractional  Separation  of  Tetanus  Bacilli    .         .         .  118,  119 


LESSON    IX 

EXAMINATION  OF  DECOMPOSING  MEAT — 
A.  Putrefaction — 

(a)  Aerobic  Putrefaction         ....  .          120 

(b}  Anaerobic  Putrefaction     ....  120,  121 

(c)  Sterilisation  and  Putrefaction    ....  121-122 


CONTENTS  xix 


PAGE 

B.  How  to  Examine  a  Sample  of  Unsound  Meat        .         .  122 

C.  Trichina  spiralis 123 

D.  Cysticercus 123 

E.  Psorosperms 124 

EXAMINATION  or  ICE  CREAMS — 

(a)  Quantitative  Examination  124,  125 

(6)  Qualitative  Examination — 

Bacillus  of  Enteric  Fever 125 

Bacterium  coli  commune  125 


LESSON    X 
EXAMINATION  OF  ANTISEPTICS  AND  DISINFECTANTS — 

A.  Method  of  Testing  Antiseptics 126 

B.  Methods  of  Testing  Disinfectants— 

(1)  Koch's  Method— 

(a)  Carbolic  Acid 128 

(b)  Mercuric  Chloride       ......          128 

(2)  Steinberg's  Method  % 129 

C.  Disinfectant  Action  of  Gases — 

(a)  Sulphur  Dioxide 129,  130 

(b)  Chlorine 131 

(c)  Ammonia 131 

LESSON    XI 

EXAMINATION  OF  AN  ANIMAL  DEAD  OF  A  BACTERIAL  DISEASE — 

(a)  Anthrax  .........          132 

(b)  Pyocyaneus  septicaemia     ......  132 

(c)  Cholera  Asiatica        .         .         .         .         .         .         .          132 

METHODS  OF  HARDENING  AND  EMBEDDING  ....    133-135 
EXAMINATION  OF  TYPHOID  SPLEEN 135 


XX  CONTENTS 


LESSON    XII 

PAGE 

TESTING  OF  FILTERS— 

A.  Filter-paper.         .         .         .         .         .         «         .         .          136 

B.  Berkefeld  Filter 136,  137 

C.  Effect  of  Use  on  a  Berkefeld  Filter  .  137,  138 


PAET  III 
BACTEEIOLOGICAL  CHEMISTEY 

LESSON    I 

PREPARATION  OF   METABOLIC   PRODUCTS   OF   MlCRO-ORGANISMS — 

(a)  Sterilisation  by  Heat         .         .         .         .         .  .           141 

(b)  Sterilisation  by  Filtration 141 

(c)  Sterilisation  by  Heat  and  Filtration  .         .         .  .  141,  142 

(d)  "  Intracellular "  Poisons  .         ...         .         .  .          142 

TEST  FOR  NITROUS  ACID  IN  CULTURES       .        .        .  .         143 

LESSON    II 

PROTEINES — 

(a)  Bacillus  pyocyaneus .         .         .     '    .         .         .  .  144,  145 

(b)  Bacillus  prodigiosus .          145 

(c)  Precipitation  by  Alcohol  .         .         .         .         .  .145,146 

BACTERIAL  EXTRACTS  146 


CONTENTS 


XXI 


LESSON    III 


BACTERIAL  PIGMENTS— 

(a)  Bacillus  pyocyaneus  . 

(b)  Bacillus  prodigiosus   . 


147,  148 
149 


PEPTONES 

ALBUMOSES — 

Proto-albumose 
Deutero-albumose 


LESSON    IV 


150 

151 
151 


LESSON   V 

ALBUMOSES  (concluded) — 
Separation  of  Albumoses 


.  152-154 


LESSON    VI 


DIPHTHERIA  ALBUMOSES— 

(a)  From  Cultures . 

(b)  From  Diphtheria  Spleen 


155-157 
157 


LESSON   VII 

DIPHTHERIA  TOXINE — 

Uschinsky's  Solution 158.  159 

ACTION  OF  MAGNESIUM  on  AMMONIUM  SULPHATE  ON  SUL- 
PHATE OF  QUININE        . 160 


xxii  CONTENTS 


LESSON    VIII 

PAGE 

FERMENTS  AND  ENZYMES — 

(a)  Ferments  and  Enzymes  in  Yeast        ....          161 
(6)  Action  of  Chloroform  on  Ferments  and  Enzymes        .  162,  163 

(c)  Action  of  Moist  Heat  on  Enzymes      .         .         .         .  163,  164 

(d)  Separation  of  Enzymes       .        .         .         ,         .         .          164 

(1)  Earth's  Method 164,  165 

LESSON    IX 

FERMENTS  AND  ENZYMES  (concluded)— 

(d)  Separation  of  Enzymes  (concluded) — 

(2)  Yon  Briicke's  Method 167,  168 

(e)  Proteolytic  Ferments  of  Micro-organisms   .         .         .          168 

(1)  Bacillus  prodigiosus    .......  168 

(2)  Bacillus  pyocyaneus 168 

(3)  Yibrio  Finkler-Prior 168 

(4)  Yibrio  choleras  Asiaticae 168 

(/)  Tryptic  Enzymes  of  Bacillus  prodigiosus  or  Bacillus 

pyocyaneus 169-171 

LESSON    X 

PTOMAINES — 

(a)  Cadaverine        .        0         ......  172-174 

(b)  Putrescine         ...  .  172-174 


PAET    I 
GENERAL    BACTERIOLOGY 

LESSONS    I-XVI 


LESSON    I 

Methods  of  Inoculation— Action  of  Light,  Oxygen,  and  Temperature 
on  the  Growth  and  Metabolism  of  Bacteria — Chromogenic  Organ- 
isms— Liquefaction  of  Gelatine. 

Methods  of  Inoculation 

SEVERAL  coloured  organisms  are  supplied  for  inoculation 
on  various  solid  media.  Students  should  come  to  some 
arrangement  between  themselves  and  divide  the  work  so  as 
to  economise  the  materials. 

Organisms  supplied — 

1.  Bacillus  prodigiosus. 

2.  Bacillus  fluorescens. 

3.  Bacillus  pyocyaneus. 

4.  Aspergillus  niger. 

5.  Sarcina  lutea. 

6.  Torula  alba. 

7.  Staphylococcus  cereus  flavus. 

8.  Staphylococcus  pyogenes  aureus. 

(1)  Make  two  agar-agar  streak  cultures  of  Bacillus  pro- 
digiosus. Place  one  in  the  warm  incubator  at  38°  0.,  and 
the  other  in  the  cool  incubator  at  20°  0. 

Examine  after  forty-eight  hours :   the  tube  kept  at 


4  GENERAL  BACTERIOLOGY  LESSON 

38°  0.  will  show  a  copious  white  growth,  while 
the  other  tube  will  show  a  pink  or  red  growth. 
Now  keep  both  tubes  at  20°  C.  The  growth 
originally  kept  at  38°  C.  may  gradually  develop 
pigment,  but  at  times  it  has  permanently  lost  its 
chromogenic  properties. 

(2)  Inoculate  two  sloped  agar-agar  tubes  with  a  single 
loop  of  a  broth  culture  of  Bacillus  pyocyaneus  (eighteen 
hours  old),  distributing  the  material  over  the  entire  surface 
of  the  agar-agar. 

Expose  one  tube  to  the  light  and  keep  the  other  at 
the  ordinary  temperature  of  the  room  protected 
from  the  light. 

Examine  them  on  successive  days  :  the  tube  exposed 
to  the  light  shows  restricted  growth,  or  perhaps  no 
growth  at  all,  in  any  case  limited  production  of 
pigment,  while  the  other  tube  will  show  a  copious 
growth,  the  culture  medium  at  the  same  time 
becoming  bright  green.  Light,  therefore,  has  an 
inhibitory  action  on  the  growth  and  activity  of 
the  Bacillus  pyocyaneus,  which  is  more  marked 
the  longer  the  rays  of  the  sun  are  allowed  to  act, 
and  the  less  material  is  used  for  inoculation. 

(3)  Repeat  the  experiment  with  the  Bacillus  prodigiosus. 
Inoculate  two  sloped  agar-agar  tubes  with  a  single  loop 

of  a  pigmented  broth  culture  of  Bacillus  prodigiosus  (about 
a  week  old),  distributing  the  material  over  the  entire 
surface  of  the  agar-agar. 

Keep  one  tube  opposite  a  sunny  window  at  the  ordinary 
temperature  of  the  room,  and  the  other  at  the  same  tem- 
perature but  protected  from  the  light. 


METHODS  OF  INOCULATION 


Growth  will  take  place  in  both  tubes,  but  the  tube 
exposed  to  light  will  show  less  pigment  than  the 
tube  kept  in  the  dark.1 

(4)  The  Bacillus  prodigiosus  requires  oxygen  for  its 
pigment  production. 

(a)  Draw  out  a  fine  capillary  pipette  and  fill  its  bulb  with 
a  small  quantity  of  a  fresh  broth  culture  of  the  Bacillus 
prodigiosus  (eighteen  hours  old).      This  is  best  done  in 
the  following  manner. 

(b)  Fuse  both  ends  of  the  freshly  made  pipette,  which  is 
naturally  sterile,  and  thrust  it  through  the  loosened  cotton- 
wool plug  into  the  culture  fluid,  breaking  its  point  by 
pushing  it  against  the  bottom  of  the  tube. 

(c)  As   the   bulb    of  the  pipette   cools   the  liquid  is 
gradually  sucked  up.     When  the  pipette  is  full,  withdraw 
it  and  again  fuse  the  broken  end. 

(d)  Now  sterilise  the  other  end  by  passing  it  several 
times  quickly  through  the  flame  and  push  it  through  the 
loosened  cotton-wool  plug  into  a  stab  gelatine  tube. 

(e)  When  it   has   cooled  down   sufficiently,   thrust  it 
through  the  gelatine  against  the  bottom  of  the  tube  in 
order  to  break  the  end  of  the  pipette. 

(/)  Then  withdraw  it  into  the  centre  of  the  gelatine 
and  apply  gentle  warmth  to  the  bulb,  until  a  minute  drop 
of  the  liquid  exudes. 

(g)  Now  allow  the  bulb  to  cool  again,  and  when  the 
liquid  has  run  back,  withdraw  the  pipette.  In  this  manner, 

1  This  is  contrary  to  the  experience  of  some  observers,  who  state 
that  the  intensity  of  pigmentation  varies  directly  with  the  amount  of 
light  supplied. 


6  GENERAL  BACTERIOLOGY  LESSON 

provided  the  pipette  was  fine  enough,  the  deeper  part  of 
the  gelatine  only  is  inoculated. 

Keep  the  tube  at  the  ordinary  temperature  of  the 

room  exposed  to  diffused  light. 

Gradually  a  whitish  growth  appears  in  the  depth  of 
the  gelatine,  which  slowly  extends  to  the  surface 
and  eventually  breaks  through.  It  will  now 
quickly  develop  its  typical  red  pigment. 

(5)  Inoculate  a  potato  tube  with  Aspergillus  niger  by 
rubbing  the  material  thoroughly  over  the  slanting  surface. 
Keep  the  tube  in  the  cool  incubator  at  20°  C. 

Examine  after  forty -eight  hours:  a  white  mould 
will  be  found  on  the  surface  of  the  potato.  Later 
on  a  few  black  spots  will  appear  on  the  white 
surface,  and  after  a  few  weeks  the  whole  growth 
will  be  black. 

(6)  Make  an  agar-agar  streak  culture  of  the  Bacillus 
fluorescens  and  keep  the  tube  at  20°  C. 

Examine  next  morning :  a  copious  growth  has 
developed  on  the  surface  of  the  agar-agar  and  the 
culture  medium  has  assumed  a  light  green  tint. 

(7)  Make  a  gelatine  streak  culture  of  Torula  alba  and 
keep  the  tube  at  20°  C. 

Examine  after  forty-eight  hours  :  there  is  no  lique- 
faction of  the  culture  medium. 

(8)  Make  a  gelatine  stab  culture  of  Sarcina  lutea  and 
keep  it  at  20°  C. 

Examine  after  forty-eight  hours :  there  is  a  slight 
yellow  growth  with  commencing  liquefaction  of 
the  gelatine. 


METHODS  OF  INOCULATION 


Examine  two  weeks  later :  the  gelatine  is  completely 
liquefied  but  clear,  the  yellow  culture  having  sunk 
to  the  bottom  of  the  tube. 
(9)  Make  a  gelatine  stab  culture — 

(a)  with  Staphylococcus  pyogenes  aureus ; 

(b)  with  Staphylococcus  cereus  flavus,  and  keep 
the  two  tubes  at  20°  C. 

Examine  after  forty  -  eight  hours  :  Staphylococcus 
pyogenes  aureus  liquefies  gelatine,  rendering  it  at 
the  same  time  turbid. 

Staphylococcus  cereus  flavus  does  not  liquefy  it. 


LESSON    II 

Types  of  Micro  -  organisms  —  Microscopic  Examination  of  Bacteria, 
stained  and  unstained — Staining  of  Micro-organisms  with  Simple 
Basic  Aniline  Dyes — Preparation  of  Cover-glass  Films. 

Types  of  Micro-organisms 

EXAMINE  microscopically  the  broth  cultures  supplied. 
Cultures  supplied — 

1.  Streptococcus  pyogenes. 

2.  Staphylococcus  pyogenes  aureus. 

3.  Bacillus  filamentosus. 

4.  Torula  alba. 

5.  Sarcina  lutea. 

(a)  Clean  a  cover-glass  (No.  1,  f  in.  by  f  in.)  with  alcohol, 
and  pass  it  through  the  flame  in  order  to  sterilise  it. 

(b)  Under   aseptic  precautions  with  a   platinum   loop 
remove  a  drop  of  the  culture  and  place  it  on  the  centre  of 
the  cover-glass. 

(c)  Gently  press  the  cover-glass  on  a  clean  slide  and 
examine  the  specimen  with  a  -^  in-  oil  immersion,  using  a 
narrow  diaphragm  and  the  concave  mirror. 

Make  sketches  of  the  various  specimens. 


LESSON  ii      STAINING  WITH  SIMPLE  ANILINE  DYES 

Staining  of  Micro-organisms  with  Simple  Basic 
Aniline  Dyes 

Examine  the  same  organisms  after  staining  with  ordinary 
aniline  dyes. 

Prepare  staining  solutions — 

(1)  To  a  watch-glass  containing  distilled  water  add 
two  to  three   drops    of    a  filtered   concentrated 
alcoholic  solution  of  fuchsine. 

(2)  To  a  watch-glass  containing  distilled  water  add  two 
to  three  drops  of  a  filtered  concentrated  alcoholic 
solution  of  gentian-violet. 

(3)  Into   a  watch-glass   filter  a  little  concentrated 
aqueous  solution  of  methylene-blue. 

(a)  Clean  cover-glasses  as  before  and  transfer  a  drop  of 
the  broth  culture  to  the  centre  of  the  cover-glass,  and  with 
the  platinum  needle  spread  it  uniformly  over  the  surface. 

(I)  Allow  the  film  to  dry  in  the  air,  covering  it  up  with 
a  watch-glass  so  as  to  protect  it  from  the  dust. 

(c)  When  dry,  pass  the  cover-glass  three  times  through 
the  flame,  holding  it  in  a  pair  of  forceps  with  the  smeared 
surface  upwards. 

(d)  Now  place  it  in  a  solution  of  20  per  cent  acetic 
acid  for  five  to  ten  minutes,  by  which  means  the  ground 
substance  is  removed  and  cleared  up. 

(e)  Wash  the  acetic  acid  off  with  distilled  water,  and 
dry  the  cover-glass  between  folds  of  filter-paper.1 

1  It  is  not  always  necessary  to  clear  the  ground  substance  with 
acetic  acid. 


10  GENERAL  BACTERIOLOGY  LESSON 

(/)  Once  more  dry  the  film  in  the  air  and  pass  it 
through  the  flame. 

(g)  Now  stain  the  specimen  by  floating  it  with  the 
film  surface  downwards  on  the  staining  solution. 

For    Streptococcus   pyogenes  and  Torula  alba   use 

fuchsine. 

For  Bacillus  filamentosus  use  methylene-blue. 
For  the  Staphylococcus  pyogenes  aureus  and  Sarcina 
lutea  use  gentian-violet. 

Leave  the  films  in  the  stain  for  some  time  (two  to 
five  minutes),  then  wash  in  water  and  dry  between  folds 
of  filter -paper ;  clean  the  unsmeared  surface,  mount  in 
water,  and  examine  under  a  high  power  and  Oc.  4, 
using  no  diaphragm,  and  the  plane  reflector.1 

If  the  specimen  is  successful,  float  it  off  the  slide,  dry 
it   again,   and    mount   it   permanently    in  xylol   balsam. 
Examine  it  with  a  ^  in.  oil  immersion. 
Make  drawings. 

If  the  specimen  is  not  sufficiently  stained,  place  it  once 
more  in  the  staining  fluid,  and  proceed  as  above. 

If  the  specimen  is  oversfcained,  it  is  better  to  prepare  a 
fresh  one.  Weak  acetic  acid  (nnnr),  however,  may  be  used 
to  decolourise  it. 

Micro-organisms  vary  greatly  in  regard  to  their  affinity 
for  dyes,  and,  again,  some  dyes  stain  more  quickly  than 
others.  Thus*  aqueous  methylene-blue  hardly  ever  over- 

1  Stained  specimens  should  always  be  examined  without  a  diaphragm, 
with  an  Abbe*  condenser  and  a  plane  reflector  ;  unstained  specimens 
with  a  narrow  diaphragm  and  a  concave  reflector  without  an  Abbe 
condenser. 


ii  STAINING  WITH  SIMPLE  ANILINE  DYES  11 

stains,  while  fuchsine  frequently  does  so.  It  is  impossible 
to  give  definite  rules  as  to  how  long  a  film  should  be  left  in 
the  stain.  It  is  safest  to  examine  the  specimen  off  and  on 
in  water,  and  if  it  be  understained,  to  continue  the  process 
of  staining. 

Sarcinae  should  be  stained  with  very  dilute  solutions, 
since  they  easily  overstain. 


LESSON  III 

Staining  of  Bacteria  (continued) — Lbffler's  Methylene-blue — Examina- 
tion of  Moulds— Hanging  Drop  Cultures. 

Lb'ffler's  Methylene-blue 

PREPARE  and  stain  cover-glass  films  of  the  various  cultures 
made  during  the  previous  lesson. 

(1)  Bacillus  prodigiosus  :  agar-agar  culture. 

(a)  On  a  clean  cover-glass  place  a  small  drop  of  sterile 
distilled  water.     With  a  sterilised  platinum  needle  remove 
a  trace  of  the  culture,  and,  mixing  it  with  the   drop  of 
water,  spread  it  uniformly  over  the  cover-glass. 

(b)  Allow  the  film  to  dry  in  the  air,  and  then  pass  the 
cover-glass  three  times  through  the  flame  and  proceed  as 
before  (vide  p.  9),  staining  the  film  with  methylene-blue. 

If  necessary,  clear  the  film  with  acetic  acid  (20  per 
cent). 

Instead  of  the  ordinary  aqueous  solution  of  methylene- 
blue  Loffler's  methylene-blue  may  be  used  with  advantage. 
This  is  one  of  the  best  and  most  certain  of  staining 
solutions,  and  gives  almost  always  good  results.  It  is 
prepared  in  the  following  manner : — 


LESSON  in  LOFFLER'S  METHYLENE-BLUE  13 

Concentrated   alcoholic   solution   of   methylene-blue, 

3  vols. 
Caustic  potash  solution  1  :  10,000,  10  vols. 

A  convenient  method  of  staining  is  to  keep  the  staining 
solution  in  a  wide-necked  glass-stoppered  pot,  and  to  hold 
the  film  in  the  staining  solution  with  a  pair  of  forceps, 
gently  moving  it  about  for  one  or  two  minutes.  Then 
transfer  it  to  water  and  wash  off  the  superfluous  stain. 

(2)  Staphylococcus  pyogenes  aureus  :  liquefying  gelatine 
culture. 

(a)  With  a  loop  remove  a  little  of  the  liquefied  gelatine 
and  spread  it  carefully  over  a  clean  cover-glass. 

(6)  Allow  the  film  to  dry  in  the  air,  and  then  pass  it 
three  times  through  the  flame. 

(c)  Remove  the  gelatine  by  means  of  acetic  acid  in  the 
usual  manner,  or  by  floating  the  film  on  warm  water  for 
five  to  ten  minutes. 

(d)  Stain  with  Loffler's  methylene-blue,  wash  in  water, 
dry  with  blotting-paper,  and  mount  in  Canada  balsam. 

Examine  with  ^-  in<  °il  immersion. 

(3)  Torula  alba  :  non-liquefying  gelatine  streak  culture. 

Proceed  in  exactly  the  same  manner  as  in  the  case  of 
Bacillus  prodigiosus  (vide  p.  12). 

Stain  with  aqueous  gentian-violet  or  aqueous  fuchsine 
solution  or  with  Loffler's  methylene-blue. 

Examine  with  -j^  in-  °il  immersion. 

Examination  of  Moulds 

Aspergillus  niger  :  potato  culture,  i 


14  GENERAL  BACTERIOLOGY  LESSON 

(a)  With  a  platinum  loop  remove  a  little  of  the  culture 
and  place  it  on  a  clean  slide. 

(b)  Add  a  drop  or  two  of  caustic  potash  (1-5  per  cent 
solution) l  and  allow  this  to  act  for  five  to  ten  minutes. 

(c)  With  filter-paper  soak  up  as  much  of  the  caustic 
potash  as  possible. 

(d)  Mount  in  Farrant's  solution,  and  examine  with  a 
low  and  a  high  power,  using  a  narrow  diaphragm. 

Make  a  drawing  of  the  fungus. 

Hanging  Drop  Cultures 

Make  hanging  drop  cultures  of 

'(1)  Streptococcus  pyogenes. 
(2)  Bacillus  filamentosus. 

(a)  Take  four  clean  hollow-ground  slides.     With  a  brush 
paint  a  ring  of  vaseline  around  the  periphery  of  the  hollow 
chambers. 

(b)  Sterilise  four  clean  cover-glasses  by  passing  them 
three  times  slowly  through  the  flame. 

(c)  Place  the  sterilised  cover-glasses  on  a  sterilised  piece 
of  wire  gauze  on  a  tripod  and  cover  it  over  with  a  glass  globe 
sterilised  by  washing  it  thoroughly  with  corrosive  sublimate 
1  :  1000.     The  wire  gauze  is  easily  sterilised  by  heating  it 
to  redness  over  a  Bunsen  flame. 

(d)  With  a  sterilised  platinum  loop  place  a  drop  of  sterile 
broth  or  liquid  gelatine  (or  agar-agar)  on  the  centre  of  each 
cover-glass,  after  allowing  the  cover-glass  to  cool. 

1  Instead  of  caustic  potash  50  per  cent  alcohol,  tQ  which  a  few 
drops  of  ammonia  have  been  added,  may  be  used. 


in  HANGING  DROPS  15 

(e)  With  a  straight  sterilised  platinum  needle  inoculate 
two  of  the  drops  with  a  minimal  trace  of  the  Streptococcus 
broth  culture,  and  two  with  a  minimal  trace  of  Bacillus 
filamentosus. 

(/)  Now  carefully  place  each  cover-glass  on  the  vaseline 
ring  around  the  hollow  on  the  slide,  the  drop  facing,  of 
course,  downwards. 

(g)  Gently  press  the  cover-glass  down  on  the  vaseline, 
so  as  to  completely  shut  off  all  air  from  the  chamber. 

(h)  Place  the  slides  in  the  warm  or  cold  incubator, 
according  as  broth  (agar-agar)  or  gelatine  is  used. 

Examine  the  hanging  drops  after  twenty-four  hours 
with  a  high  power,  using  a  narrow  diaphragm. 
In  the  one  case  there  will  be  a  pure  culture  of 
streptococci,  in  the  other  a  pure  culture  of  filament 
ous  bacilli. 

Examination  of  Bacteria  in  a  Hanging  Drop 

To  study  the  motility  of  micro-organisms,  hanging  drops 
should  be  made. 

(a)  Take  an  ordinary  clean  slide. 

(b)  With  a  cork-borer  cut  a  small  ring  -|-  to  |j-  in.  in 
diameter  out  of  filter-paper,  four  to  eight  layers  thick. 

(c)  Trim  the  perforated  filter-paper  so  as  to  fit  the  slide, 
moisten  it  with  water,  and  place  it  on  the  centre  of  the 
slide. 

(d)  With  a  sterilised  platinum  loop  place  a  drop  of  a  fresh 
broth  culture  of  the  Bacillus  filamentosus  on  the  centre  of 
a  clean  sterilised  cover-glass. 


16  GENERAL  BACTERIOLOGY  LESSON  in 

(e)  Place  the  cover-glass  drop  downwards  over  the  central 
perforation  of  the  moistened  filter-paper. 

Examine  the  drop  with  a  high  power,  using  a  narrow 
diaphragm. 

Focus  for  the  margin  of  the  drop,  and  then  move  the 
specimen  till  a  bacillus  appears  in  the  field. 

Now  place  a  drop  of  oil  on  the  cover-glass,  and  examine 
with  a  -^  in.  oil  immersion. 

Distinguish  between  true  and  Brownian  movements. 


LESSON  IV 

Staining  of  Hanging  Drop  Cultures— Gram's  Method  of  Staining— 
Staining  of  Pus — Curdling  Ferment. 

Staining  of  Hanging  Drop  Cultures 

Bacillus  filamentosus. 

(a)  Carefully  remove  the  cover-glass  and  allow  the  drop 
culture  to  dry  in  the  air. 

(b)  Wipe  off  as  much  of  the  vaseline  as  possible. 

(c)  Pass  the  film  three  times  through  the  flame. 

(d)  Carefully  stain  in  Loffler's  methylene-blue  for  two 
to  three  minutes. 

(e)  Wash  very  gently  in  water. 

(/)  Dry  the  film  between  folds  of  filter-paper,  and  when 
it  is  quite  dry  mount  in  xylol  balsam. 

Examine  it  with  a  high  power,  and  then  with  -^  ifi- 
oil  immersion.  Observe  the  beautiful  network  of 
filaments. 

Gram's  Method  of  Staining 

Streptococcus  pyogenes. 

Stain    the    hanging    drop    cultures    of    Streptococcus 
pyogenes  by  this  method. 

C 


18  GENERAL  BACTERIOLOGY  LKSSON 

Solutions  required : — 

(a)  Aniline  Gentian-Violet  is  prepared  in  the  .following 
manner  :: — 

(a)  First  make  aniline  water  by  shaking  up  4  cc.  of  aniline 
oil  with  100  cc.  of  distilled  water  for  one  "to"  two  minutes. 

(b)  Filter  the  resulting  emulsion  through  filter- paper 
moistened  with  distilled  water. 

(c)  To  100  cc.»of  aniline  water  add  11  cc.  of  a  concen- 
trated alcoholic  solution  of  gentian-violet.  . 

Shake  the  mixture  thoroughly  and  alicays  filter  it  before  use. 

This  solution  does  not  keep  well,  and  should  therefore 
be  prepared  in  small  quantities. 

(y8)  Gram's  Iodine  Solution  :• — 
Iodine  crystals 

Potassium  iodide     .  .  .2  grammes 

Distilled  water         .  .  /  300  cc.  4 

(y)  Absolute  Alcohol 
Method  of  staining  : — 

(a)  Prepare  the  hanging  drop  culture  of  the  Streptococcus, 
as  described  before  for  Bacillus  filamentosus,  and  when  the 
film  is  dry  and  has  been  passed  through  the  flame,  place  it 
first  in  alcohol  for  one  to  two  minutes. 

(b)  Without  drying  it,  transfer  it  to  the  aniline  gentian- 
violet,  and  leave  it  in  the  stain  for  half  a  minute  to 'one 
minute. 

(c)  Soak  up  the  superfluous  stain  with  blotting-paper. 

(d)  Now  place  it  in  Gram's  solution  of  iodine  for  half  a 
minute  to  one  minute. 


iv  GRAM'S    METHOD  19 

(e)  Soak  up  the  superfluous  iodine  solution,  and  gently 
wash  in  absolute  alcohol,  until  no  more  stain  comes  away. 

(/)  Quickly  wash  the  alcohol  off  in  water,  and  dry  the 
film  between  folds  of  blotting-paper. 

(g)  When  the  cover-glass  is  quite  dry,  mount  it  in  xylol 
balsam. 

Examine  with  a  high  power,  and  then  with  -^  in.  oil 
immersion :  long  chains  of  Streptococci  will  be 
seen,  stained  dark  violet. 

Prepare  Cover-glass  Films  of  a  Liquefied  Culture  of  Sta- 
phylococcus  Pyogenes  Aureus,  and  Stain  by  Gram's 
Method. 

(a)  Prepare  the  film  and  pass  it  through  acetic  acid  in 
the  usual  manner  (vide  p.  9). 

(&)  Wash  off  the  acetic  acid  with  water,  dry  the  film 
and  pass  it  three  times  through  the  flame. 

(c)  Now  place  the  film  in  absolute  alcohol  for  one  to 
two  minutes,  ano!  stain  with  aniline  gentian -violet,  and 
continue  as  above  with  Gram's  solution,  alcohol,  etc. 

Staining  of  Pus 

(1 )  With  methylene-llue. 
{2)  By  Gram's  method. 

(a)  With  a  platinum  loop  smear  a  thin  film  of  pus  on 
two  clean  cover-glasses. 

(b)  Allow  the  films  to  dry  in  the  air,  and  then  pa^them 
three  times  through  the  flame,  keeping  the  smeared  surface 
upwards. 


20  *$      GENERAL  BACTERIOLOGY  LESSON 

(c)  Place  the  films  in  20  per  cent  acetic  acid  for  three  to 
five  minutes. 

(d)  Wash   them  in  water,  dry  them  between  folds  of 
blotting-paper,  and  pass  them  again  through  the  flame.    The 
films  are  now  ready  for  staining. 

(1)  Lqffler's  methylene-blue. 

(a)  Place  one  film  in  the  stain  for  two  to  five  minutes. 

(b)  Wash  in  water,  dry  and  mount  in  xylol  balsam. 

Examine  with  ^  in.  oil  immersion  :  observe  the 
leucocytes  and  cocci  (Staphylococci  or  Streptococci, 
or  both ;  many  probably  in  the  leucocytes). 

(2)  Gram's  method. 

(a)  Place  the  other  film  in  absolute  alcohol  for  one  to 
two  minutes. 

(b)  Stain  it  in  aniline  gentian-violet  for  one  to  two  minutes. 

(c)  Remove  the  superfluous  stain  with  blotting-paper. 

(d)  Now  place  in  Gram's  solution  of  iodine  for  half  a 
minute,  until  the  specimen  turns  black. 

(e)  Soak  up  the  superfluous  iodine  solution. 

(/)  Wash  in  alcohol  till  the  film  is  almost  colourless. 

(g)  Pass  quickly  through  a  diluted  alcoholic  solution  of 
cosine,  which  stains  the  leucocytes  and  ground  substance 
pink. 

(h)  Wash  in  water,  dry  and  mount  in  xylol  balsam. 

Examine  with  a  high  power,  and  then  with  -fa  m-  °^ 
immersion  :  the  pus  cocci  are  stained  dark  violet, 
and  also  the  chromatine  of  the  nuclei ;  the  ground 
substance  and  protoplasm  of  the  leucocytes  are  pink. 


rv  CURDLING    FERMENT 

Prepare  Cultures  of  Bacillus  Anthracis 

From  the  agar-agar  culture  supplied  inoculate — 

(1)  a  potato  tube  ; 

(2)  a  broth  tube ; 

(3)  an  agar-agar  tube  (streak) ; 

(4)  a  gelatine  tube  (stab). 

Place  (1)  (2)  and  (3)  in  the  warm  incubator,  and  (4)  in 
the  cool  incubator. 

Examine  after  forty-eight  hours  :  observe  the  charac- 
teristic growths  in  the  broth  and  gelatine.  The 
latter  is  slowly  liquefied. 

Curdling  Ferment 

Some  micro-organisms  will  coagulate  milk,  others  will 
not. 

Inoculate  four  milk  tubes  with — 

(1)  Bacillus  fluorescens ; 

(2)  Bacterium  coli  commune  ; 

(3)  Bacillus  anthracis ; 

(4)  Bacillus  prodigiosus. 

Place  the  inoculated  tubes  in  the  warm  incubator. 

Examine  the  tubes  after  forty-eight  hours :  the  milk 
in  (1)  and  (2)  will  be  coagulated,  that  in  (3)  and 
(4)  may  or  may  not  be. 


LESSON  V 

Bacillus  Anthracis— Mode  of  Growth  on  Various  Media— Asporogenous 
Cultures — Acid  Formation  by  Virulent  Anthrax  Bacilli— Methods 
of  Attenuation — Impressions  (Sloped  Gelatine) — Methods  of 
Staining. 

Bacillus  Anthracis 

(1)  EXAMINE  the  tubes  inoculated  with  Bacillus  anthracis 
and  note  the  mode  of  growth  on  the  various  media, 

(2)  Prepare  a  streak  culture  of  Bacillus  anthracis   on 
neutral  litmus   agar-agar,    and    place    it    in    the    warm 
incubator. 

Examine  it  after  forty-eight  hours,  and  notice  the 
change  in  the  colour  of  the  litmus.  The  colour 
gradually  assumes  a  reddish  tint  on  account  of 
the  acid  produced  by  the  bacillus. 

(3)  Prepare   two   hanging   drop   cultures  (broth,  agar- 
agar,  or  gelatine)  of  Bacillus  anthracis  (vide  p.  14),  and 
place  them  in  the  incubator. 

(4)  Inoculate   a    broth   tube    containing    carbolic   acid 
(1  : 1000),  and  keep  it  at  38°  C. 

Examine  it  microscopically  a  week  later :  no  spores 
will  be  found  (asporogenous  growth). 


LESSON  v  BACILLUS    ANTHRACIS  23 

(5)  Inoculate   a  broth   tube  and  place   it  in  a  water 
incubator  at  42*5°  C. 

Examine  it  microscopically  a  week  later  :  no  spores 
will  be  found,  and  the  bacillus,  moreover,  is 
attenuated  and  less  virulent. 

(6)  Inoculate  two  neutral  litmus  agar-agar  tubes :  one 
with   ordinary  virulent   bacilli   and   the   other  with   the 
attenuated  bacillus. 

The  latter  produces  less  acid. 

(7)  (a)  Inoculate  a  tube  containing  a  little  sterilised  *75 
per  cent  saline  solution  with  three  platinum  loops  of  an 
agar-agar  culture  of  Bacillus  anthracis. 

(b)  Shake  the  inoculated  tube  vigorously,  and  from  it, 
with  a  platinum  loop,  inoculate  a  sloped  gelatine  tube, 
thoroughly  streaking  the  surface  of  the  culture  medium. 

(c)  Place  the  tube  in  the  cool  incubator,  and  when  small 
colonies   appear   they  are  to  be  worked  up  by  means  of 
impression  specimens  (vide  infra  p.  26). 

(8)  Staining  of  Bacillus  anthracis. 

(a)  Prepare  cover-glass  specimens  in  the  usual  manner — 

(1)  of  a  broth  culture  of  Bacillus  anthracis ; 

(2)  of  a  gelatine  culture ; 

(3)  of  an  agar-agar  culture. 

(b)  Stain  films— 

(a)  With  Loffler's  methylene-blue  after  having  cleared 
with  acetic  acid. 

(/3)  With  gentian-violet  after  having  cleared  with  warm 
water. 


24  GENERAL  BACTERIOLOGY  LESSON  v 

(7)  According  to  Gram's  method  (vide  p.  18). 

Examine  with  a  high  power  and  also  with  -^  in.  oil 
immersion :  bacilli  and  spores  will  be  found,  the 
spores  remaining  unstained. 

(9)  Prepare  a  hanging  drop  of  a  broth  culture  of  the 
Bacillus  anthracis,  and  examine  it  with  -^  m«  oil  immersion. 
The  Bacillus  anthracis  is  not  motile. 


LESSON  VI 

Bacillus  Antliracis  (continued) — Hanging  Drop  Cultures — Methods  of 
Staining  Impressions — Examination  of  Fresh  Tissues  by  means 
of  Cover -glass  Films — Staining  of  Leucocytes  for  Eosinophile 
Granules. 

Bacillus  Antliracis  (continued] 

(I)  EXAMINE  the  litmus  agar-agar  tube  inoculated  with 
Bacillus  anthracis,  and  note  the  gradual  disappearance  of 
the  blue  or  purple  colour. 

(2)  Examine  the  hanging  drop  cultures  with  a  high 
power. 

Make  a  drawing. 

Stain  (a)  one  of  them  with  Loffler's  methylene-blue  in 
the  usual  manner  (vide  p.  13);  (b)  the  other  according  to 
Gram's  method. 

(3)  Prepare  a  cover -glass   specimen  of  the  culture  in 
carbolic  acid  broth  and  stain  it  with  Loffler's  methylene- 
blue,  or  according  to  Gram's  method. 

Note  the  absence  of  spores. 

(4)  Inoculate  a  litmus  agar-agar  tube  from  the  broth 
culture  kept  at  42-5°  C.,  and  another  litmus  agar-agar  tube 
from  an  ordinary  broth  culture  grown  at  38°  C. 


26  GENERAL  BACTERIOLOGY  LESSON 

Place  the  former  in  the  water  incubator  at  42 '5°  C., 
and  keep  the  latter  at  38°  C. 

Compare  them  after  forty-eight  to  eighty  hours  and 
notice  the  difference  in  the  tint  of  the  litmus.  The 
latter  or  more  virulent  culture  decolourises  the 
agar-agar  more  rapidly. 

Impression  Specimens 

(a)  Examine  the  sloped  gelatine  tubes  (vide  Lesson  V.  7, 
p.   23)  with  a   magnify  ing -glass  for  colonies  of  anthrax 
bacilli. 

(b)  If  found,  rapidly  dip  the  gelatine  tube  into  boiling 
water,   having  previously  removed  the  cotton-wool  plug. 
This  momentary  heating  will  free  the  gelatine  from  the 
sides  of  the  tube. 

(c)  Slide  the  gelatine  block  out  on  to  a  cool  black  glass 
plate  with  the  culture  surface  upwards,  and  trim  the  sides  of 
the  gelatine  block  with  a  sharp  scalpel  dipped  in  methylated 
spirit. 

(d)  Select  small  superficial  colonies  and  gently  press  a 
clean  cover-slip  down  over  them. 

(e)  Now   carefully   remove   the    cover- glass  with   the 
impression  of  the  colonies  adhering  to  its  under  surface. 

(/)  Gently  warm  the  cover-glass  and  then  pass  it  three 
times  through  the  flame. 

(g)  Stain  with  Loffler's  methylene-blue,  or  according  to 
Gram's  method,  and  mount. 

Examine  under  a  high  power,  and  make  a  drawing  of 
the  impression. 


vi  EXAMINATION   OF    FKESH    TISSUES  27 

Examination  of  Fresh  Tissues  by  means  of  Cover-Glass  Films 

Examine  the  spleen  of  the  white  mouse  dead  of 
anthrax,  for  the  presence  of  Bacillus  anthracis,  by  means 
of  cover-glass  specimens. 

(a)  Pin  the  animal  out  on  a  small  board  washed  with 
sublimate  solution  (roVo)- 

(b)  Moisten  the  hair  on  the  abdomen  and  chest  with 
methylated  spirit. 

(c)  Carefully  reflect  the  skin  of  the  abdomen  and  chest. 

(d)  Heat  a  small  glass  rod  to  redness  in  the  blow-pipe, 
and  rub  the  surface  of  the  abdomen  with  it,  especially 
along  the  linea  alba,  and  on  each  side  along  the  costal 
arches. 

(e)  With  a  sterilised  pair  of  forceps  and  scissors  carefully 
open  the  abdominal  cavity  in  the  middle  line,  make  two 
transverse  cuts  along  the  costal  arches,  and  throw  the  flaps 
outwards. 

(/)  With  a  fresh  pair  of  sterilised  forceps  and  scissors 
remove  a  small  piece  of  the  swollen  spleen  and  gently 
smear  it  over  a  clean  cover-glass.  Prepare  four  films  in 
this  way. 

(g)  Allow  them  to  dry  in  the  air,  and  then  pass  them 
three  times  through  the  flame. 

(h)  Stain  them  according  to  the  following  methods : — 

(1)  In  methylene-blue  without  previously  passing  the 
film  through  acetic  acid. 

(2)  First  pass  the  film  through  acetic  acid  in  the  usual 
manner  and  then  stain  with  Loffler's  methylene-blue. 


28  GENERAL  BACTERIOLOGY  LESSON  vi 

(3)  Stain  a  film  with  eosine  and  methylene-blue. 

(a)  Dip  the  cover-glass  for  a  few  seconds  in  a  wide- 
necked   pot   containing    *5   per   cent   solution   of 
eosine  in  50  per  cent  alcohol. 

(b)  Wash  it  in  water,  and  dry  it  with  blotting-paper. 

(c)  Pass  it  three  or  four  times  through  the  flame,  and 
now  stain  quickly  in  Loffler's  methylene-blue. 

This  method  stains  the  eosinophile  granules  of  the 
leucocytes  and  also  brings  out  the  red  corpuscles. 

(4)  Stain  a  film  according  to  Gram's  method,  first  passing 
the  specimen  through  acetic  acid.    Counterstain  with  eosine. 

In  each  case  mount  in  xylol  balsam  and  examine  with 
•£%  in.  oil  immersion. 

Note  the  absence  of  spores  and  the  relation  of  the 
bacilli  to  the  leucocytes. 


LESSON  VII 

Bacillus  Anthracis  (continued} — Plate  Cultures — Staining  of  Spores- 
Separation  of  Bacillus  Subtilis  from  Hay  Infusion. 

Bacillus  Anthracis  (continued) 

EXAMINE  the  litmus  agar-agar  cultures  of  Bacillus  anthracis 
made  on  the  last  occasion. 

Note  the  difference  in  tint  of  the  two  tubes. 

Plate  Culture  Method 

(a)  Take  a  broth  culture  of  Bacillus  anthracis  (forty- 
eight  hours  old)  and  three  gelatine  tubes. 

(6)  Liquefy  the  gelatine  tubes  in  the  warm  incubator. 

(c)  Shake  the  broth  culture,  and  from  it  inoculate  the 
first  gelatine  tube  with  three  platinum  loops,  and  label  it  I. 

(d)  From  I.,  after  shaking,  inoculate  the  second  gelatine 
tube  with  three  platinum  loops,  and  label  it  II. 

(e)  From  II.  similarly  inoculate  the  third  tube,  and  label 
it  III. 

(/)  Take  three  sterilised  Petri's  capsules  and  place  them 
on  moist  filter-papers,  and  label  them  I.  II.  and  III. 

(g)  Now  heat  the  mouths  of  I.  II.  and  III.  in  the  flame 
and  allow  them  to  cool. 


30  GENERAL  BACTERIOLOGY  LESSON 

(h)  Pour  out  each  gelatine  tube,  after  gently  shaking  it, 
into  the  corresponding  capsule. 

(i)  Allow  the  gelatine  to  set  in  a  uniform  layer,  and 
when  set,  place  the  capsules  in  the  cool  incubator. 

Examine  on  the  next  occasion :  colonies  will  appear 
on  each  plate,  naturally  most  numerously  on  plate 
I.  and  least  on  plate  III.  (vide  infra  p.  34). 

Staining  of  Spores 

Bacillus  filamentosus,  or  anthracis,  or  megatherium  (agar- 
agar  or  potato  cultures). 

Solutions  required : — 

(a)  Ehrlich's,  Loffler's,  or  Ziehl's  fuchsine. 

(/3)  Acid  alcohol :  alcohol  97  cc.,  hydrochloric  acid  3  cc. 

(7)  Loffler's  methylene-blue. 

EhrlicKs  fuchsine :  Aniline  water  100  cc. 

Concentrated    alcoholic    solution    of 
fuchsine  11  cc. 

This  staining  solution  does  not  keep  well,  and  there- 
fore only  small  quantities  should  be  made  at  a 
time. 

Loffler's  fuchsine :  Aniline  water  100  cc. 

Fuchsine  crystals  4  to  5  grammes. 

1  per  cent  solution  of  caustic  soda  1  cc. 

Shake  till  dissolved  (about  half  an  hour). 


vii  SPORE    STAINING  31 

Ziehl's  fuchsine :  5  per  cent  aqueous  solution  of  carbolic 

acid  100  cc. 

Concentrated     alcoholic     solution     of 
fuchsine  11  cc. 

All  these  solutions  must  be  filtered  before  use. 

First  Method. 

(a)  Prepare  a  film  in  the  usual  manner  and  pass  it  three 
times  through  the  flame. 

(5)  Filter  some  of  the  fuchsine  solution  into  a  small 
evaporating  dish  and  gently  warm  it,  keeping  it  just 
steaming. 

(c)  Float  the  cover-glass,  film  surface  downwards,  on  the 
warm  stain  and  continue  gentle  warming  for  twenty  to 
thirty  minutes. 

(d)  Now  wash  the  specimen  in  water. 

(e)  Dip  it  for  a  few  seconds  in  the  acid  alcohol,  and  at 
once  wash  it  again  in  water  to  remove  the  acid. 

(/)  Examine  it  in  water,  under  a  high  power,  for  spores, 
which  should  appear  as  round  or  oval  red  bodies,  the 
bacilli  themselves  being  faintly  pink  or  unstained. 

If  the  spores  are  unstained  or  too  much  decolourised, 
place  the  film  back  in  the  fuchsine  solution,  and 
decolourise  for  a  shorter  time  in  the  acid  alcohol. 

If,  on  the  other  hand,  the  bacilli  are  not  sufficiently 
decolourised,  dip  the  cover-glass  once  more  into 
the  acid  alcohol. 

(g)  When  the  spores  are  well  stained,  and  the  bacilli 
practically  unstained,  dry  the  specimen  with  blotting-paper 
and  pass  it  three  times  through  the  flame. 


'      3?  GENERAL  BACTERIOLOGY  LESSON 

(h)  Now  counferstam  with  Loffler's  methylene-blue,  wash 
in  water,  dr^  and  mount. 

Examine  with  ^  ^n-  °^  immersion :  the  bacilli  are 
Jilue  and  the  spores  red. 

Make  a  drawing. 

The  spores  of  the  Bacillus  filamentosus  and  the 
Bacillus  megatherium  are  more  easily  stained  than 
those  of  the  Bacillus  anthracis. 

Second  Memod. 

(a)  Prepare  a  film  in  the  usual  manner. 

(b)  Pass  it  three  times  through  the  flame.  * 

(c)  Place  it  for  two  mrftutes  in  absolute  alcohol,  and  for 
further  two  minutes  in  chloroform. 

(d)  Wash  the  film  in  water,  dry  it  between  blotting- 
paper,  and  pass  it  again  three  times  through  the  flame. 

(e)  Leave  it  for  two  minutes  in  a  5  per  cent  solution  of 
chromic  acid,  and  then  wash  it  in  water,  and  dry  it  between 
blotting-paper. 

(/)  Once  more  pass  ^^tree  times  through  the  flame,  and 
place  it  in  a  warm  solution  of  carbol  fuchsine  for  five  to 
fifteen  minutes. 

(g)  Rinse  the  film  in  water,  and  decolourise  in  5  per  cent 
sulphuric  acid,  till  the  specimen  is  faintly  pink. 

(h)  Wash  it  in  water  and  counterstain  in  malachite 
green. 

(i)  Wash  again,  dry  and  ftiount. 

t 
The  spores  are  red  and  well  stained,  the  bacilli  green. 


VII 


SEPARATION  OF  THE  HAY  BACILLUS 


Separation  of  the  Hay  Bacillus  (Bacillus  siibtilis) 
out  of  Hay  Masion. 

Six  tubes  containing  hay  infusion  are  suppliec^ 
Heat  them  for  five*  ;ten,  fifteen,  twenty,  twenty-five  and 
thirty  minutes  respectively  in  water  at  -80°  C. 

From   each  tube   inoculate  a   broth  Hibe  with   three 
platinum  loops  of  the*  infusion  and  label  them. 

Keep  the  six  tribes  in  the  cool  incubator  and  examine 
them  on  the  next  occasion. 

Pure  cultures  of  the  hay  bacillus  will  probably  be 
obtained. 


LESSON  VIII 

Bacillus  Anthracis  (concluded)— Impression  Specimens  (Gelatine  Plates) 
—Staining  of  Anthrax  Bacilli  in  Tissues— Staining  of  Flagella. 

Bacillus  Anthracis  (concluded] 

WITH  a  dissecting  microscope  examine  the  plates,  made  on 
the  previous  occasion,  for  colonies  of  anthrax  bacilli. 

Observe  the  characteristic  appearance  of  the  colonies. 

Now  place  a  clean  cover-glass  over  a  colony  and  examine 
it  with  a  high  power. 

Make  a  drawing. 

Impression  specimens. 

(a)  Take  a  perfectly  clean  cover-glass,  sterilise  it  by  pass- 
ing it  through  the  flame,  and  place  it  over  a  suitable  colony. 

(b)  With  a  needle  carefully  press  the  cover-glass  down, 
until  the  colony  is  slightly  flattened  out. 

(c)  With  a  sterilised  needle  lift  the  cover-glass  with  the 
colony  adhering  to  it  from  the  surface  of  the  gelatine ;  then 
proceed  as  described  before  (vide  p.  26). 


LES.  vin     STAINING  OF  ANTHRAX  BACILLI  IN  TISSUES     35 , 


Staining  of  Tissues  for  Anthrax  Bacilli 
(Frozen  Sections) 

(1)  Simple  staining  with  Lo'ffler's  methylene-blue. 

Tissues  supplied :  lung,  kidney,  liver  and  spleen  of  a 
mouse  dead  of  anthrax. 

(a)  Place  a  section  in  Loffler's  methylene-blue  for  five 
minutes. 

(b)  Wash  it  in  distilled  water  for  half  a  minute. 

(c)  Then  decolourise  it  for  a  quarter  to  half  a  minute 
with  very  dilute  acetic  acid  (one  to  two  drops  of  glacial 
acetic  acid  to  a  watch -glassful  of  water),  till  the  section 
is  pale  blue. 

(d)  Again  wash  it  in  water  for  half  a  minute. 

(e)  Now  place  it  in  absolute  alcohol  for  half  a  minute  to 
one  minute. 

(/)  Clear  the  section  in  xylol.1 

(g)  Transfer  the  specimen  to  a  slide  with  a  piece  of 
cigarette-paper. 

(h)  Mount  it  in  xylol  balsam. 

Examine  it  with  low  and  high  powers  and  y1^   in. 
oil  immersion. 

(2)  Double  staining  with  methylene-blue  and  cosine. 
Prepare  the  following  solution  (Czinzinski's  solution) : — 

1  Instead  of  xylol  other  clearing  media  may  be  used,  as  bergamot 
oil,  a  mixture  of  turpentine  and  creasote,  or  cedar-wood  oil. 


36  GENERAL  BACTERIOLOGY  LESSON 

Concentrated  aqueous  solution  of 

methylene-blue      .          .          .50  cc. 
Eosine  .....        -5  gramme 
Absolute  alcohol        .  .      70  cc. 

Distilled  water          .          .          .    130  cc. 

(a)  Keep  the  section  in  absolute  alcohol  for  five  minutes. 

(b)  Transfer  it  to  the  stain  for  four  to  twelve  hours. 

(c)  Wash  it  in  water  till  the  section  appears  pink,  and 
shows  hardly  any  trace  of  blue. 

(d)  Dehydrate,  clear,  and  mount  in  the  usual  manner. 

Examine  with  low  and  high  powers.     The  bacilli  are 
stained  blue  and  the  tissues  pink. 

(3)  Double  staining  with  picrocarmine  and  Loffler's  methylene- 
blue. 

(a)  Place  the  section  in  alcohol  for  half  a  minute. 

(b)  Then  wash  it  in  water  and  place  it  in  a  solution  of 
picrocarmine  and  water  (equal  parts)  for  ten  to  twenty 
minutes. 

(c)  Wash  it  in  spirit,  to  which  a  few  drops  of  l^dro- 
chloric  acid  have  been  added,  for  half  a  minute  to  one 
minute. 

(d)  Wash  it  in  water. 

(e)  The  sections  are   now  stained  red,   and  should  be 
placed  in  methylene-blue  and  treated  as  described  before. 

(4)  Staining  of  sections  by  Gram's  method. 

(a)  Place  the  section  in  alcohol  for  half  a  minute. 


viii  CULTIVATION  OF  VIBRIOS  37 

(b)  From  the   alcohol  transfer    it   directly   to   filtered 
aniline  gentian-violet  and  allow  it  to  stain  for  two  to  ten 
minutes. 

(c)  Einse  the  section  in  distilled  water. 

(d)  Now  place  it  in  Gram's  iodine  solution  till  it  is 
black  (about  two  minutes). 

(e)  Transfer  it  to  absolute  alcohol  for  a  quarter  to  half 

a  minute,  and  thence  to 

0f* 
(/)  Acid  alcohol  (alcohol  with  3  per  cent  hydrochloric 

acid)  for  not  more  than  ten  seconds. 

(g)  Again  wash  it  in  alcohol  until  the  section  appears 
colourless,  or  if  previously  stained  with  picrocarmine,  until 
the  section  again  becomes  red. 

(h)  Clear  it  in  xylol  or  turpentine  and  creasote. 
(i)  Transfer  it  to  a  slide  with  a  cigarette-paper. 
(k)  Mount  it  in  Canada  balsam. 

Examine  with  low  and  high  powers  and  -^  in.  oil 
immersion :  the  bacilli  appear  dark  blue  on  a 
yellowish  or  red  background. 

Cholera 

Inoculate — 

(1)  a  gelatine  tube  (stab),  an  agar-agar  tube  (streak), 
and  a  broth  tube  with  Vibrio  cholerse  Asiaticse  ; 

(2)  a  gelatine  tube  (stab),  an  agar-agar  tube  (streak), 
and  a  broth  tube  with  Vibrio  Finkler-Prior ; 

(3)  a  gelatine  tube  (stab),  an  agar-agar  tube  (streak), 
and  a  broth  tube  with  Vibrio  Metchnicovi. 


38  GENERAL  BACTERIOLOGY  LESSON 

Place  the  gelatine  tubes  in  the  cool  incubator  at  22°  C., 
the  others  in  the  warm  incubator  at  38 -5°  C. 

Staining  of  Flagella  (Van  Ermengem) 

Prepare  the  following  solutions : — 

(a)  Osmic  Acid  (2  per  cent  solution)  1  part. 
Tannin  (10  to  25  per  cent  solution)  2  parts. 

To  each  100  cc.  of  the  tannin  solution  add  four  or 
five  drops  of  glacial  acetic  acid. 

(/3)  Nitrate  of  silver  (-25  to  -5  per  cent  solution). 

(y)  Gallic  Acid  ...  5  grammes. 
Tannin  ...  3  grammes. 
Fused  acetate  of  soda  .  10  grammes. 
Distilled  water  .  .350  cc. 

Boil  the  cover-slips  to  be  used  in  the  following  solution : — 

Potassium  bichromate  .         60  grammes. 

Concentrated  Sulphuric  Acid         60  grammes. 
Water     ....     1000  cc. 

Then  wash  them  repeatedly  in  water.  Keep  them  in 
absolute  alcohol  and  before  use  allow  them  to  dry,  without 
wiping,  by  placing  them  in  a  vertical  position,  protected 
from  dust.  •* 

Bacillus  of  typhoid  fever  and  Vibrio  cholem  Asiatic®. 

Carefully  suspend  one  or  two  loops  of  an  agar-agar  culture 
(ten  to  eighteen  hours  old)  in  a  watch-glassful  of  distilled 
water. 


STAINING  OF  FLAGELLA 


(a)  With  a  single  loopful  of  this  "  suspension  "  prepare 
a  cover-glass  film  and  allow  it  to  dry  in  the  air. 

(6)  Fix  it  by  passing  it  three  times  through  the  flame, 
holding  the  specimen  in  the  fingers,  so  as  to  avoid  over- 
heating. 

(c)  Pour  'a  few  drops  of  solution  (a)  on  the  film  and 
allow  them  to  act  for  half  an  hour.1 

(d)  Wash  very  carefully  in  a  large  excess  of  distilled 
water,  and  then  in  alcohol. 

(e)  Now  keep  it  for  three  to  five  seconds  in  solution  (/3). 
(/)  Without  washing,  pass  quickly  through  solution  (<y). 
(g)  Wash  again   in  a  fresh   quantity  of  solution  (/9), 

moving  the  specimen  about  gently  and  .withdrawing  it  when 
the  solution  begins  to  turn  black. 

(h)  Wash  it  thoroughly  in  several  changes  of  distilled 
water. 

(i)  Dry  it  carefully  between  blotting-paper.  • 

Mount  it  first  in  water  and  examine  it  with  -^  in.  oil 
immersion,  and  if  the  specimen  be  satisfactory, 
mount  it  permanently  in  xylol  balsam. 

If  the  flagella  are  not  sufficiently  stained,  float  the  film 
off  the  slide  and  begin  again  at  (/). 

Care  must  be  taken  to  change  the  nitrate  of  silver  solu- 
tion as  soon  as  any  precipitation  shows  itself. 

This  is  an  easy  and  very  trustworthy  method. 
1  At  a  temperature  (K  60°  C.  five  minutes  Is  sufficient. 


LESSON  IX 

Examination  of  Vibriones  Choleras  Asiaticse,  Finkler  -  Prior,  and 
Metchnicovi  in  Cultures  and  in  the  Animal  Body — Plate  Cultures 
— Germicidal  Action  of  Sunlight — Hanging  Drops. 

Cholera  (continued) 

(1)  EXAMINE  and  compare  the  tubes  inoculated  with 
Vibrio  cholerse  Asiaticse,  Vibrio  Finkler-Prior,  and  Vibrio 
Metchnicovi  respectively. 

(2)  Prepare  cover -glass  specimens  of  Vibrio  choleras 
Asiaticse,  Vibrio  Finkler-Prior,  and  Vibrio  Metchnicovi  in 
the  usual  manner. 

Stain  them  with  dilute  aqueous  gentian- violet  or  dilute 
aqueous  fuchsine,  as  follows  : — 

(a)  Filter  some  of  the  staining  solution  into  a  watch- 


(5)  Stain    for   about    five   to   ten   minutes,    previously 
clearing  the  specimen  with  acetic  acid,  if  necessary. 

(c)  Wash  in  water,  dry  and  mount. 

Examine  with  ^  in.  oil  immersion  and  compare  the 
three  forms. 


LESSON  ix  EXAMINATION  OF  VIBRIOS  41 

(3)  Prepare  cover-glass  specimens  of  Spirillum  rubrum 
(gelatine  stab  culture). 

(a)  Melt  the  gelatine  tube  in  the  warm  incubator. 

(b)  Prepare  films  and  pass  them  three  times  through  the 
flame  in  the  usual  manner. 

(c)  Wash  the   films   in  warm   water  for  two   to   five 
minutes,  in  order  to  remove  the  gelatine. 

(d)  Stain    in    dilute    gentian  -  violet    or    fuchsine,    as 
above. 

(e)  Wash  in  water,  dry  and  mount. 

Examine  with  -^  in.  oil  immersion. 

(4)  From    a    liquefied    gelatine    culture    of    Spirillum 
rubrum  prepare  a  gelatine   stab  culture  and  keep  it  at 

22°  C. 

(5)  Prepare  hanging   drop  cultures  of   Vibrio  cholerae 
Asiatics,  using  1  per  cent  peptone  solution. 

Place  them  in  the  warm  incubator. 

(6)  Examine  the  peritoneal  fluid  of  the  guinea-pig  supplied 
for  Vibrio  cholerce  Asiaticce. 

(a)  Open  the  abdominal  cavity  of  the  animal  in  the  usual 
manner  (vide  p.  27). 

(b)  Make  a  capillary  pipette,  and  draw  up  some  of  the 
peritoneal  fluid  into  its  bulb. 

(c)  Prepare  films  from  the  fluid  removed,  in  the  usual 
manner,  and  pass  them  three  times  through  the  flame. 

(d)  Clear  them  with  acetic  acid  and  then  stain  with 
aqueous  gentian-violet,  as  already  described  (vide  supra). 

Examine  with  -      in.  oil  immersion. 


42  GENERAL  BACTERIOLOGY  LESSON 

__ — 

(e)  Other  films  should  be  stained  with  cosine  and  methy- 
lene-blue,  without  previously  clearing  them  with  acetic 
acid  (vide  p.  28). 

Examine  with  -^  in.  oil  immersion  :  note  the  eosino- 
phile  granules  in  the  leucocytes. 

Plate  Cultures  of  Vibrio  Choleras  Asiatics 

Open  the  thorax  (of  a  guinea-pig  inoculated  intra- 
peritoneally  with  the  Vibrio  cholerae  Asiaticae)  in  the 
following  manner : — 

(a)  Moisten  the  skin  with  spirit. 

(b)  Reflect  it  freely  on  both  sides. 

(c)  Cauterise  the  exposed  surface,  and  writh  a  pair  of 
sterilised  scissors  and  forceps  remove  the  sternum. 

(d)  Now  cauterise  with  a  heated  glass  rod  a  spot  on  the 
surface  of  the  heart. 

(e)  Thrust  a  sterile  capillary  pipette,  the  ends  of  which 
have  been  broken  off,  through  the  heart's  wall  into   an 
auricle  or  ventricle  and  remove  a  trace  of  blood  by  suction, 
and  fuse  the  end  to 'which  suction  has  been  applied. 

(/)  Loosen  the  cotton-wool  plug  of  a  liquefied  gelatine 
tube  and  insert  the  pipette  into  the  test-tube  by  carefully 
pushing  it  through  the  cotton-wool. 

(g)  Push  the  pipette  into  the  lumen  of  the  tube,  till  it 
almost  touches  the  gelatine. 

(h)  Apply  very  gentle  heat  to  the  bulb,  till  a  drop  of 
blood  falls  into  the  gelatine. 

(i)  Withdraw  the  pipette  and  shake  the  inoculated  tube. 


ix  EFFECT  OF  SUNLIGHT  ON  BACTERIA  43 

(k)  From  this  tube  inoculate  two  other  tubes  in  the 
ordinary  manner,  and  pour  three  plates  as  described  in  a 
former  lesson  (vide  p.  29). 

Keep  the  plates  at  22°  C.  and  examine  them  from  day 
to  day. 

Action  of  Sunlight  on  the  Vibrio  Cholerse  Asiaticae 

To  demonstrate  the  action  of  sunlight  on  cholera  vibrios 
take  two  agar-agar  plates  (Petri's  capsules)1  which  have 
been  kept  in  the  warm  incubator  for  twelve  to  twenty-four 
hours. 

With  a  sterilised  brush  gently  paint  the  surface  of 
the  agar-agar  with  a  dilute  young  broth  culture  of  cholera 
vibrios. 

Over  the  lid  of  each  capsule  gum  a  piece  of  black  paper 
with  a  large  figure  or  letter  cut  in  it,  and  expose  the  plates 
to  the  sun  for  four  to  six  hours. 

Then  transfer  the  plates  to  the  warm  incubator. 

Examine  them  on  the  next  day :  there  will  be  no 
growth  (or  only  a  limited  growth)  on  the  agar-agar 
over  those  areas  which  were  unprotected  from  the 
action  of  the  light  by  the  black  paper. 

1  For  preparation  of  agar-agar  plates  see  Part  II. 


LESSON   X 

Vibrio  Cholerse  Asiatics  (concluded) — Impression  Specimens  from  Plates 
—  Comparison  of  Different  Varieties  —  Curdling  Ferment  — 
Actinomycosis — Cladotlirix — Staining  of  Tissues  embedded  in 
Celloidin— Flagella  Staining  after  Pitfield. 

Cholera  (concluded) 

(1)  EXAMINE  the  plates  made  from  the  heart's  blood  of 
the  guinea-pig. 

The  colonies  may  be  so  small  that  they  can  only  be  seen 
with  a  magnifying-glass. 

Select  a  typical  colony  and  make  a  gelatine  stab  culture 
from  it  which  should  be  kept  at  22°  C. 

Prepare  also  impression  specimens  of  suitable  colonies, 
and  stain  them  with  aqueous  gentian-violet. 

Examine  with   a   high   power   and   T^   in.    oil   im- 
mersion. 

(2)  Inoculate  five  gelatine  tubes  from  the  five  varieties 
of  cholera  vibrios  supplied  (stab  cultures). 

Place  them  in  the  cool  incubator  at  22°  C. 

Compare  their  mode  of   growth  and  rate  of  lique- 
faction from  day  to  day. 


LESSON  x      STAINING  OF  CELLOIDIN  SECTIONS  45 

(3)  Inoculate  five  milk  tubes  with  the  same  varieties 
of  cholera  vibrios,  and  keep  them  at  38 '5°  C. 

Compare  their  mode  of  growth  and  curdling  power 
from  day  to  day 

(4)  Examine  the  hanging  drop  cultures  of  Vibrio  cholerse 
Asiatics  previously  made,  and  stain  them  with  aqueous 
gentian-violet. 

Actinomyces  and  Cladothrix 

(1)  Inoculate  an  agar-agar  tube  with  Cladothrix  nivea 
and  another  with  Cladothrix  asteroides. 

Keep  them  in  the  cool  incubator  at  22°  C. 

(2)  Stain  actinomycotic  tissue,   embedded  in  celloidin,   by 
Gram's  method. 

Some  of  the  sections  may  be  previously  stained  with 
picrocarmine  (vide  p.  36). 

(a)  Place  the  section  on  a  slide  and  with  a  sharp  scalpel 
trim  off  the  celloidin  all  round. 

(b)  Dry  the  specimen  with  filter-paper. 

(c)  Filter  a  little  aniline  gentian-violet  on  to  the  section, 
and  cover  it  over  with  a  watch-glass,  so  as  to  protect  it 
from  the  dust. 

(d)  After  a  quarter  to  half  an  hour  remove  the  gentian- 
violet   with    blotting-paper    (sometimes   five    minutes    is 
sufficient). 

(e)  Pour  a  little  of  Gram's  iodine  solution  on  the  section 
and  allow  it  to  act  for  one  to  two  minutes. 

(/)  Remove  the  iodine  solution  with  I lotting-paper. 


46  GENERAL  BACTERIOLOGY  LESSON  x 

(g)  Pour  a  little  aniline  oil  over  the  specimen,  which  is 
now  quite  black,  and  by  alternately  tilting  first  one  and 
then  the  other  end  of  the  slide,  allow  the  oil  to  flow  to 
and  fro  over  the  section.  The  aniline  oil  will  take  up  the 
gentian-violet  greedily,  and  gradually  become  saturated. 
When  this  has  taken  place,  remove  the  oil  with  blotting- 
paper  and  pour  fresh  oil  over  the  specimen.  Repeat  this 
process  till  no  more  stain  comes  away. 

(h)  Eemove  the  oil  with  blotting-paper,  and  wash  the  speci- 
men thoroughly  with  xylol,  which  must  be  frequently  changed. 
(i)  Mount  in  xylol  balsam  and  examine  with  low  and 
high  powers. 

It  is  essential  that  the  whole  process  be  completely 

carried  through  with  the  section  on  the  slide. 
Sections    of    Mycetoma   (Madura   disease)   may   be 
stained  in  the  same  manner. 

Staining  of  Flagella  (after  Pitfield) 

(1)  Prepare  the  following  solution  : —    ^~  ) 

(a)  Cold  saturated  solution  of  alum  10  cc.  Saturated 
alcoholic  solution  of  gentian-violet  2  cc.ft  ) 

To  this  add  (&)  10(cc.  of  a  cold  aqueous  solution  of 
tannic  acid  (10  per  cent). 

(2)  Prepare  a  suspension  of  typhoid  bacilli,  cover-slips 
and  films  as  on  pp.  38  and  39. 

(3)  Cover  the   film  with   the   stain,  and   keep   gently 
steaming  on  a  copper  section  lifter  for  1-2  minutes. 

(4)  Wash  in  water,  dry  and  mount. 

Examine  with  ^-  in.  oil  immersion.  The  results  are 
almost  always  excellent,  and  remarkably  easily 
obtained. 


LESSON   XI 

Pyogenic  Cocci — Cultures — Hanging  Drops — Staining  of  Pus  preserved 
in  Carbolic  Acid — Staining  of  Fresh  Pus— Staining  of  Gonorrhoeal 
Pus. 

Pyogenic  Cocci 
(1)  MAKE  a  broth  culture  of  the 

(a)  Streptococcus  pyogenes ; 
(I)  Streptococcus  pneumonias  ; 
(c)  Streptococcus  erysipelatos. 

Keep  them  at  3 8 '5°  C.,  and  compare  them  from  day 
to  day. 

(2)  Make  also  agar-agar  streak  cultures   of   the   same 
organisms. 

Keep  them  at  the  same  temperature,  and  compare 
them  from  day  to  day. 

(3)  Make  gelatine  stab  cultures  of  the  same  organisms 
and  keep  them  at  22°  C. 

They  all  grow  slowly  in  gelatine. 
Compare  them  from  day  to  day. 

(4)  Prepare  agar-agar  streak  cultures  of  the  Staphylo- 
coccus  pyogenes  albus,  aureus,  and  cereus  flavus  respec- 
tively, and  keep  them  at  38*5°  C. 


48  GENERAL  BACTERIOLOGY  LESSON 

(5)  Prepare  gelatine  streak  cultures  of   the  Staphylo- 
coccus  pyogenes  aureus  and  cereus  flavus  respectively,  and 
keep  them  at  22°  C. 

The  former  will  liquefy  the  gelatine,  the  latter  will 
not. 

(6)  Prepare  two  hanging  drop  cultures  of  the  Strepto- 
coccus pyogenes  in  broth,  and  keep  them  at  38'5°  C. 

(7)  Prepare  films  from  the  condensation  water  of  an 
agar-agar    culture    of    the    Streptococcus    pyogenes,   and 
stain  them 

(a)  with  gentian-violet,  fuchsine,  or  Loffler's  methy- 
lene-blue ; 

(b)  by  Gram's  method. 

Mount  in  xylol  balsam  and  examine  with  a  high 
power,  or  ^  in-  °il  immersion. 

(8)  Prepare  specimens  of  the  pus  supplied  which  con- 
tains Streptococci.     The  pus  has  been  preserved  in  carbolic  acid 
(5  per  cent  solution). 

(a)  With  a  platinum  loop  remove  some  of  the  white 
sediment  and  place  it  on  a  clean  cover-glass. 

(b)  Place  another  cover-glass  on  this  and  rub  the  two 
cover-glasses  together  so  as  to  spread  the  films  uniformly. 

(c)  Separate  the  cover-glasses  and  allow  the  two  films 
to  dry. 

(d)  When  dry,  pass  the  films  three  times  through  the 
flame. 

(e)  Place  the  films  in  a  mixture  of  alcohol  and  ether 
(equal  parts)  for  one  to  two  minutes. 


xi  PUS  AND  GONORRHCEA  49 


(/)  Dry  them  between  blotting-paper  and  again  pass 
them  three  times  through  the  flame. 

(g)  Now  stain  them  in  the  usual  manner  with  dilute 
aqueous  gentian-violet,  or  by  Gram's  method. 

Mount  in  xylol  balsam  and  examine  with  -£%  in.  oil 
immersion. 

(9)  Prepare  specimens  of  the  iresh  pus  supplied  which 
contains  Staphylococci. 

Prepare  thin  films  in  the  usual  manner,  clear  them  in 
acetic  acid,  and  stain  with  cosine  and  methylene-blue  in 
the  following  way  (vide  p.  28) : — 

(a)  Stain  with  cosine. 

(b)  Wash,  and  dry  with  filter-paper. 

(c)  Heat  again  and  stain  with  Loffler's  methylene-blue. 

(d)  Wash,  dry  and  mount. 
Examine  with  ^  in-  °^  immersion. 

(10)  In  the  same  manner  prepare  and  stain  specimens  of 
gonorrhoeal  pus.     (Eosine  and  methylene-blue  or  gentian- 
violet.) 

Examine  with  T^  in.  oil  immersion :  note  the  cocci 
in  the  cells. 

Stain  another  specimen  by  Gram's  method,  counter- 
staining  with  eosine,  and  contrast  with  previous  specimens. 

The  gonococci  do  not  retain  the  stain. 


LESSON  XII 


Pyogenic  Cocci  (concluded) — Staining  of  Hanging  Drops — Staining  in 
Tissues — Fibrin  Staining  in  Croupous  Pneumonia. 


Pyogenic  Cocci  (concluded) 

(1)  STAIN  the  hanging  drop  cultures  of  the  Streptococcus 
pyogenes  prepared  on  the  previous  occasion — 

(a)  with  dilute  fuchsine  ; 

(b)  by  Gram's  method. 

Mount  in  xylol  balsam  and  examine  with  a  high 
power. 

(2)  Examine  unstained  the  broth  cultures,  of  the  three 
Streptococci,  made  last  time. 

(a)  Shake  the  tube,  and  with  a  sterilised  loop  remove  a 
drop  of  the  fluid,  and  place  it  on  a  clean  cover-glass. 

(b)  Carefully  place  the  cover-glass  on  a  clean  slide  and 
with  a  brush  paint  some  vaseline  round  the  margin  of  the 
cover-glass,  so  as  to  prevent  evaporation. 

Examine  with  a  high  power  and  ^  in.  oil  immersion, 
using  a  narrow  diaphragm. 


LESSON  xii        STAINING  OF  COCCI  IN  TISSUES  51 

(3)  Stain  frozen  sections  of  the  following  tissues : — 

(a)  Pysemic  abscesses  in  cardiac  muscle  and  kidney 
(Staphylococci  from  a  case  of  ulcerative  endocarditis). 

Use  Loffler's  methylene-blue,  or  Gram's  method. 

Place   some   sections   in  the   cosine   methylene-blue 
mixture  (Czinzinski's  solution)  over  night. 

*   (b)  Erysipelatous  skin. 

Use  Gram's  method,  with  or  without  previous  picro- 
carmine  staining. 

(c)  Spleen  from  a  case  of  pyaemia  (Streptococcus). 
Use  Gram's  method  without  counterstaining. 

(d)  Croupous  pneumonia  (lung). 

Use  Gram's  method  with  or  without  previous  picro- 
carmine  staining. 

(e)  Lung  of  mouse  dead  of  infection  with  the  Micro- 
coccus  tetragonus. 

Stain  with  the  cosine  methylene-blue  mixture. 

In  all  these  cases,  whenever  Gram's  method  is  used, 
leave  the  section  in  the  stain  for  twenty  to  thirty  minutes, 
and  be  careful  not  to  decolourise  too  much  with  the  alcohol 
and  acid  alcohol  (vide  p.  45). 

(4)  Fibrin  staining  in  croupous  pneumonia  (Weigert's  method). 

To  bring  out  the  fibrin  network  in  croupous  pneumonia, 
the  following  method  should  be  used  : — 

(a)  Place  the  section  on  the  slide  by  means  of  a  cigarette- 
paper. 


52  GENERAL  BACTERIOLOGY  LESSON  xn 

(6)  Eemove  the  superfluous  water  with  a  piece  of  filter- 
paper. 

(c)  Filter  a  little  aniline  gentian-violet  on  to  the  section, 
and  allow  it  to  act  for  twenty  to  thirty  minutes. 

(d)  Eemove   the   aniline   gentian-violet   with   blotting- 
paper,  and  wash  with  *6  per  cent  saline  solution. 

(e)  Pour  a  few  drops  of  the  following  iodine  solution  on 
to  the  section  : — 

Iodine       .  1  gramme 

Potassium  iodide  .          .  2  grammes 

Water        ....          100  cc. 

Allow  this  to  act  for  two  to  three  minutes. 

(/)  Eemove  the  iodine  solution  with  blotting-paper, 
and  decolourise  with  a  solution  of  aniline  oil  (2  parts)  and 
xylol  (1  part),  in  exactly  the  same  manner  as  described  in 
the  case  of  actinomycosis  (vide  p.  46). 

(g)  Wash  thoroughly  with  xylol  to  remove  all  trace  of 
the  aniline  oil. 

(h)  Mount  in  xylol  balsam  and  examine  with  a  high 
power. 

The  fibrin   network  and   the   Pneumococci  will  be 
distinctly  shown  in  most  cases. 

To  make  the  specimen  more  effective  stain  the  section 
previously  with  picrocarmine. 


LESSON   XIII 

Bacillus  of  Typhoid  Fever  and  Bacterium  Coli  Commune — Cultures 
and  Differences  iii  Mode  of  Growth — Shake  Cultures — Varieties  of 
Bacterium  Coli  Commune — Staining  of  Typhoid  Spleen — Staining 
of  Capsule  of  Pneumococcus — Diphtheria — Staining  of  Diphtheria 
Bacilli — Staining  of  Diphtheritic  Membrane — Staining  of  Leprosy 
Bacillus  in  the  Tissues. 

Bacillus  of  Typhoid  Fever  and  Bacterium  Coli  Commune 

(1)  PREPARE   cultures   of   these  two   organisms  for  com- 
parison : — 

(a)  Gelatine,  streak  and  stab  (at  22'5°C.) 

(b)  Potato  (at  38-5°  C.) 

(c)  Agar-agar  streak  (at  38 '5°  C.) 

(d)  25  per  cent  gelatine,  streak  and  stab  (at  38*5°  C.) 

(e)  Broth  (at  38*5°  C.) 
(/)  Milk  (at  38-5°  C.) 

(g)  Gelatine  containing  4  per  cent  of  a  5  per  cent 
solution  of  carbolic  acid  (at  22°  C.) 

Compare  these  cultures  from  day  to  day  :  the 
Bacterium  coli  coagulates  milk,  the  typhoid 
bacillus  does  not ;  the  typhoid  bacillus  produces 
rapid  turbidity  in  25  per  cent  gelatine,  the 


54  GENERAL  BACTERIOLOGY  LESSON 

• 

Bacterium  coli  much  more  slowly;  in  other  respects 
the  two  organisms  in  cultures  resemble  each 
other  closely.  (For  Indol  Reaction  vide  p.  100.) 

(2)  Prepare  "  shake  cultures  "  of  these  two  organisms. 

(a)  Melt  two  ordinary  gelatine  tubes,  and  inoculate  one 
with  the  Bacterium  coli  and  the  other  with  the  Bacillus 
typhosus. 

(&)  Shake  the  tubes  and  allow  them  to  set. 
(c)  Keep  them  at  22°  C. 

Examine  them  next  day :  very  active  gas  formation 
takes  place  in  the  gelatine  in  the  case  of  the 
Bacterium  coli  commune,  none  in  the  case  of  the 
typhoid  bacillus. 

(3)  Prepare  films  of  these  two  organisms  in  the  usual 
manner. 

Stain  with  aqueous  gentian-violet  for  one  to  two 
minutes,  wash  in  water,  dry  and  mount. 

Examine  with  T^-  in.  oil  immersion,  and  compare  the 
two  organisms. 

There  are  several  varieties  of  the  Bacterium  coli  commune. 

(4)  Prepare  agar-agar  shake  cultures  of  Varieties  I.,  II., 
and  III. 

(a)  Melt  the  agar-agar  either  by  placing  it  in  boiling 
water    or  by   carefully   heating   the   tubes   over   a   small 
Bunsen  flame,  and  allow  the  tubes  to  cool  to  40°  C. 

(b)  Now  inoculate  them  with  Varieties  I.,  II.,  and  III. 
respectively. 


xm        STAINING  OF  CAPSULE  OF  PNEUMOCOCCUS  55 

(c)  Allow  the  agar-agar  to  set,  and  keep  the  tubes  at 
38-5°  C. 

Compare  them  from  day  to  day  for  gas  formation. 

(5)  Inoculate  three  milk  tubes  with  the  same  Varieties, 
and  keep  them  at  38 '5°  C. 

Examine  them  from  day  to  day  for  coagulation. 
Curdling  may  be  rapid  or  delayed  or  even  absent. 

(6)  Typhoid  Spleen  (vide  also  p.  135). 

Stain  sections  of  typhoid  spleen  with  Loffler's  methylene- 
blue  or  in  Czinzinski's  fluid,  in  the  usual  manner. 

Examine  with  a  low  and  high  power,  and  -£%  in. 
oil  immersion. 

(7)  To  stain  the  capsule  of  the  Pneumococcus  prepare  the 
following  solution  : — 

Concentrated   alcoholic  solution  of 

gentian-violet          .           .  .  5  cc. 

Distilled  water  .          .          .  .  10  cc. 

Glacial  acetic  acid        .           .  .  1  cc. 

(a)  In  this  solution  stain  films,  prepared  from  pneumonic 
sputum,  for  twenty-four  hours. 

(b)  Then  wash  in  acetic  acid  (1  per  cent)  for  one  or  two 

minutes. 

(c)  Wash  in  water. 

(d)  Dry  between  blotting-paper. 

(e)  Mount  in  xylol  balsam. 

Examine  with  -      in.  oil  immersion. 


56  GENERAL  BACTERIOLOGY  LESSON 

(8)  Stain  pneumonic  sputum  kept  in  carbolic  acid  ly 
Weigerfs  method,  counterstaining  with  cosine. 

(a)  Prepare  thin  films,  as  previously  described,  and  after 
having  passed  them  three  times  through  the  flame,  wash 
them  in  the  alcohol-ether  mixture  (vide  p.  48). 

(b)  Dry  the  films,  and  again  pass  them  through  the  flame. 

(c)  Stain  them  with  aniline  gentian-violet  for  fifteen  to 
thirty  minutes. 

(d)  Wash  them  in  *6  per  cent  saline  solution. 

(e)  Place  them  in  Weigert's  iodine  solution  (iodine  1, 
pot.  iod.  2,  water  100)  for  two  to  three  minutes. 

(/)  Dry  thoroughly  with  filter-paper,  and  pour  a  weak 
alcoholic  solution  of  eosine  over  the  cover-glass,  and  allow 
this  to  act  for  thirty  seconds  or  less. 

(g)  Quickly  wash  in  *6  per  cent  saline  solution. 

(h)  Dry  again  with  filter-paper,  and  decolourise  with  the 
anilme-xylol  solution  till  the  specimen  is  pink. 

(i)  Wash  with  xylol  arid  mount  in  xylol  balsam. 
Examine  with  -^  in-  oil  immersion. 

If  the  sputum  has  not  been  kept  in  carbolic  acid, 
prepare  thin  films  of  the  fresh  viscid  sputum,  and  after 
passing  the  dry  films  three  times  through  the  flame,  at  once 
proceed  to  stain  \vith  the  aniline  gentian-violet,  and  continue 
as  above. 

Diphtheria 

(1)  Inoculate  a  serum  agar-agar  tube  (streak),  and 
a  gelatine  tube  (streak)  from  an  agar-agar  culture.  Keep 


xiii  DIPHTHERIA  AND  LEPROSY  57 

the   serum   tube   at   38-5°    C.  and   the   gelatine   tube   at 
22°  C. 

(2)  Prepare   cover-glass   specimens   from   an   agar-agar 
culture  and  stain  with  aniline  gentian-violet. 

(a)  Leave  them  in  the  stain  for  five  to  ten  minutes. 

(b)  Decolorise  in  spirit. 

(c)  Wash  in  water,  dry  and  mount. 

Examine  with  ^  in-  °^  immersion  :  note  the  clubbed 
forms  and  the  characteristic  grouping. 

(3)  Diphtheritic  membrane. 

Stain  sections  of  diphtheritic  membrane  with  the 
eosine  methylene-blue  mixture  in  the  usual  manner  (vide 
p.  35). 

Examine  with  low  and  high  powers. 

(4)  Prepare  hanging  drop  cultures  in  broth  and  keep 
them  at  38  -5°  C. 

Examine  them  from  day  to  day,  so  as  to  study  the 
peculiar  forms  which  the  diphtheria  bacillus  often 
shows. 

Leprosy  Tissue 

(1)  Stain   with    picrocarmine,    and   then  according   to 
Weigert's  fibrin  method  (vide  p.  51). 

Examine  with  low  and  high  powers  and  ^  in-  °il 
immersion,  and  note  the  arrangement  of  the 
bacilli  and  their  relation  to  the  cells. 

(2)  Stain  a  section  with  a  filtered  carbol  fuchsine  solution. 


58  GENERAL  BACTERIOLOGY  LESSON  xin 

. _ 

(a)  Leave  in  the  stain  for  twenty  to  thirty  minutes. 
,  (b)  Wash  it  quickly  in  -6  per  cent  saline  solution. 

(c)  Decolourise  it  in  hydrochloric  acid  (25  per  cent)  for 
a  few  seconds,  until  the  red  tint  disappears. 

(d)  Transfer  the  section  to  70  per  cent  alcohol,  and  with 
a  needle  move  it  about  till  no  more  red  is  washed  out. 

(e)  Remove  the  alcohol  with  '6  per  cent  saline  solution. 

(/)  Now  counterstain  the  section  in  Loffler's  methylene- 
blue  for  one  to  two  minutes. 

(g)  Wash  thoroughly  in  -6  saline  solution. 

(h)  Dehydrate  in  absolute  alcohol. 

(i)  Clear  in  xylol  and  mount  in  xylol  balsam. 

Examine  with  low  and  high  powers  and  -^  in-  oil 
immersion.  The  bacilli  are  stained  red  and  the 
tissues  blue. 


LESSON  XIV 

Tubercle  Bacillus— Staining  of  Fresh  Sputum— Staining  of  Carbolised 
Sputum— Staining  of  Frozen  Sections— Staining  of  Paraffin  Sections. 

(1)  EXAMINE  the  cultures  made  on  the  last  occasion. 

(2)  Prepare  films  of  the  recent  cultures  of  the  Bacillus 
typhosus  and  the  Bacterium  coli  commune  and  stain  them 
with  methylene-blue. 

(3)  Prepare  films  of  the  fresh  culture  of  the  diphtheria 
bacillus,  and  stain  them  with  aniline  gentian-violet  (vide 


Tubercle  Bacillus 

(1)  Staining  of  fresh  sputum. 

(a)  Pour  out  the  sputum  in  a  thin  layer  into  a  glass  dish, 
and  placing  the  latter  on  a  dark  background  select  one  of 
the  characteristic  yellowish  particles,  and  pick  it  out  with  a 
pair  of  fine-pointed  forceps. 

(6)  Prepare  thin  films  by  squeezing  the  suspected  matter 
between  two  clean  cover-glasses. 

(c)  Allow  them  to  dry  in  the  air,  and  then  pass  them 
three  times  through  the  flame. 


GENERAL  BACTERIOLOGY 


(d)  Float  the  films  on  a  warm  carbol  fuchsine  solution 
for  two  to  five  minutes. 

(e)  Take  one  of  them  out,  leaving  the  other  in  the  stain 
for  subsequent  examination  if  necessary. 

(/)  Wash  it  rapidly  in  water  to  remove  the  excess  of 
fuchsine. 

(g)  Decolourise  it  in  25  per  cent  hydrochloric  acid  by 
holding  the  cover-glass  with  a  pair  of  forceps  and  dipping 
it  in  the  acid  just  long  enough  to  discharge  the  red  colour. 

(h)  Wash  it  immediately  in  60  to  70  per  cent  spirit :  at 
first  the  red  colour  reappears ;  continue  to  wash  till  no  more 
red  comes  off. 

(i)  Wash  it  in  water  to  remove  the  spirit. 

(k)  Dry  the  film  between  filter-paper  and  pass  it  again 
three  times  through  the  flame. 

(I)  Now  stain  it  in  Lofner's  methylene-blue  (ten  to 
twenty  seconds). 

(m)  Wash  it  once  more  in  water. 

(n)  Dry  it  between  filter-paper  and  again  pass  it  through 
the  flame  to  dry  it. 

(o)  Mount  it  in  xylol  balsam  and  examine  it  with  an  oil 
immersion. 

The  tubercle  bacilli  are  stained  red  on  a  blue  ground. 

i 
If  no  tubercle  bacilli  be  found,  the  film  which  was 

left  in  the  stain  should  be  treated  and  examined 
in  the  same  manner. 

It  is  best  to  keep  the  water,  acid,  spirit,  and  methylene- 
blue  in  wide -necked  glass -stoppered  pots.  Holding  the 


xiv  STAINING  OF  TUBERCULOUS  SPUTUM  61 

cover-glass  in  a  pair  of  forceps,  pass  it  successively  from 
one  pot  to  another,  a  procedure  which  saves  both  time  and 
material.  For  the  solutions  are  always  ready  for  use  and 
may  be  employed  over  and  over  again. 

Repeat  the  process,  using  carbol  gentian-violet  instead 
of  carbol  fuchsine,  and  vesuvine  or  Bismarck-brown  instead 
of  methylene-blue. 

Carbol  gentian-violet : — 

5  per  cent  carbolic  acid  .  .10  vols. 

Concentrated    alcoholic   solution   of 

gentian-violet  ...        1  vol. 

Leave  the  specimen  in  this  stain,  which  must  be  warmed 
of  course,  for  at  least  five  minutes. 

Be  careful  not  to  leave  the  film  in  the  hydrochloric 
acid  too  long. 

Vesuvine  should  be  used  in  concentrated  aqueous 
solution. 

The  tubercle  bacilli  are  stained  blue  on  a  brown  ground. 

(2)  Sputum  kept  in  carbolic  acid.  \ 

(a)  Pour  100  cc.  of  5  per  cent  carbolic  acid  into  a  small 
flask. 

(b)  Add  10  to  20  cc.  of  the  sputum,  unless  the  latter  is 

watery  (vide  infra). 

(c)  Shake  vigorously  ^or  five  minutes,  till  the  sputum  is 
thoroughly  disintegrated. 

(d)  Pour  the  contents  of  the  flask  into  a  conical  urine- 
glass  and  allow  it  to  stand  for  twelve  to  twenty-four  hours. 

(e)  Decant  the  supernatant  fluid. 


62  GENERAL  BACTERIOLOGY  LESSON 

(/)  With  a  fine  pipette  remove  a  little  of  the  sediment 
from  the  extreme  depth  of  the  glass  and  rub  it  between 
two  cover-glasses. 

(g)  Dry  the  films  in  the  air  and  pass  them  three  times 
through  the  flame. 

(h)  Wash  them  in  a  mixture  of  alcohol  and  ether  (equal 
parts)  for  one  to  three  minutes. 

(i)  Dry  them  between  filter-paper  and  again  pass  them 
three  times  through  the  flame. 

(k)  Stain  them  with  carbol  fuchsine  or  carbol  gentian- 
violet,  and  proceed  in  the  manner  described  above. 

This  is  by  far  the  best  method  for  detecting  tubercle 
bacilli. 

If  the  sputum  is  watery,  pour  100  cc.  of  it  into  a  flask 
and  add  5  cc.  of  liquefied  carbolic  acid,  shake  for 
five  minutes,  and  then  proceed  as  above. 

Urine  suspected  of  containing  tubercle  bacilli  must  be 
treated  like  watery  sputum. 

(3)  Staining  of  frozen  sections  of  tuberculous  tissues. 
(i.)  Quick  Method. 

(a)  Place  the  sections  in  carbolic  acid  (5  per  cent)  for 
one  to  two  hours.     (This  may  be  omitted.) 

(b)  Stain  them  in  carbol  fuchsine  or  carbol  gentian-violet 
for  two  hours  in  an  incubator  at  3  8 '5°  0. 

(c)  Wash  a  section  rapidly  in  '6  per  cent  saline  solution 
or  in  distilled  water. 

(d)  Now  transfer  it  to  25  per  cent  hydrochloric  acid, 
and  leave  it  till  the  colour  changes  to  yellow  or  brown :  a 
few  seconds  are  sufficient. 


xiv  STAINING  OF  TUBERCULOUS  TISSUES  63 

(e)  Wash  it  in  70  per  cent  spirit,  till  no  more  stain 
comes  away. 

(/)  Again  wash  the  section  in  distilled  water,  or  '6  per 
cent  saline  solution. 

(g)  Counterstain  it,  for  two  minutes  in  Loftier' s  methylene- 
blue  if  carbol  fuchsine  was  used,  or  in  vesuvine  for  five 
minutes  if  carbol  gentian- violet  was  employed. 

(h)  Wash  it  thoroughly  in  distilled  water. 

(i)  Dehydrate  it  in  absolute  alcohol  and  clear  it  in 
xylol. 

(k]  Transfer  the  section  on  to  a  slide  and  mount  it  in 
xylol  balsam. 

Examine   it    with  a   high   power    and    ^   in.    oil 
immersion. 

(ii.)  Slow  Method. 

(a)  Place  a  section  in  aniline  fuchsine  or  aniline  gentian- 
violet  for  twelve  to  twenty-four  hours. 

(b)  Wash  it  rapidly  in  water  and  decolourise  it  in  25  per 
cent  hydrochloric  acid,  and  then  proceed  as  above. 

This  method  gives  extremely  good  results, 
(iii.)  Gram's  Method. 

(a)  Stain  a  few  sections  first  with  picrocarmine  in  the 
ordinary  manner  (vide  p.  36). 

(b)  Place  them  in  aniline  gentian-violet  for  twenty-four 
hours. 

(c)  Wash  a  section  rapidly  in  water. 


G4  GENERAL  BACTERIOLOGY  LESSON 

(d)  Place  it  in  Gram's  iodine  solution  for  one  to  two 
minutes. 

(e)  Wash  it  in  absolute  alcohol  for  half  a  minute  and 
then  in  acid  alcohol  (3  per  cent  hydrochloric  acid)  for  not 
longer  than  eight  to  ten  seconds. 

(/)  Again   wash   it   in   absolute   alcohol   till  no  more 
stain  comes  off,  and  the  specimen  appears  red. 

(g)  Dehydrate  it  in  absolute  alcohol. 

(h)  Clear  it  in  xylol  and  mount  it  in  xylol  balsam. 

Examine  it  with  a  high  power  and  T^  in.  oil  im- 
mersion. The  bacilli  are  stained  blue  on  a  red 
ground. 

Staining  of  Paraffin  Sections 

(1)  Tubercle. 
(i.)  Slow  Method. 

(a)  Fix  a  section  on  a  clean  cover-glass  (if  possible  with- 
out egg  albumen  or  any  other  fixing  medium)  and  remove 
the  paraffin  in  the  usual  manner  (heat,  xylol,  alcohol,  and 
water). 

(b)  Float  the  cover-glass,  specimen  surface  downwards, 
on  aniline  fuchsine  for  twenty  to  twenty-four  hours,  or  on 
carbol  fuchsine  for  two  to  three  hours. 

(c)  Now  pass  it  through  water,  hydrochloric  acid,  spirit, 
water,  and  methylene-blue  in  exactly  the  same  manner  as 
was  done  in  the  case  of  sputum  films. 

(d)  Wash  it  in  water,  dehydrate  in  absolute  alcohol, 
clear  in  xylol,  and  mount. 


xiv  STAINING  OF  PARAFFIN  SECTIONS  65 

Aniline  gentian-violet  or  carbol  gentian- violet  may  be 
used. 

This  method  gives  by  far  the  best  results,  the  sections 
being  thin  and  well  stained,  and  it  should  be 
employed  in  all  cases  where  good  specimens  are 
required.  The  paraffin  must  be  thoroughly  re- 
moved. 

(ii.)  Quick  Method. 

(a)  Fix  a  section  on  a  clean  cover-glass,  and  remove  the 
paraffin  as  before. 

(b)  Now  float  the  specimen  on  the  surface  of  a  warm 
carbol  fuchsine  solution  for  five  to  ten  minutes,  and  treat 
it  as  if  it  were  an  ordinary  sputum  film,  except  that  it 
must  be  dehydrated  and  cleared  before  mounting. 

The  section  must  be  as  thin  as  possible,  and  the  carbol 
fuchsine  solution  should  not  be  overheated.  This 
method,  if  combined  with  quick  paraffin  em- 
bedding, is  of  great  value  when  a  rapid  diagnosis 
is  desirable. 

(2)  Diphtheritic  membrane. 

(a)  Fix  a  section  of  the  diphtheritic  membrane  on  a 
cover-glass  in  the  usual  manner  and  remove  the  paraffin. 

(b)  Place  the  cover-glass  in  the  eosine  methylene-blue 
mixture  for  six  to  eighteen  hours. 

(c)  Wash  it  in  water  till  the  blue  tint  has  almost  dis- 
appeared. 

(d)  Dehydrate  it  in  absolute  alcohol,  clear,  and  mount 
it  in  xylol  balsam. 


56  GENERAL  BACTERIOLOGY  LESSON  xiv 

Stain  another  section  according  to  Weigert's  fibrin 
method  : 

(a)  Place  it  in  aniline  gentian-violet  for  half  an  hour  to 
one  hour. 

(b)  Wash  it  in  -6  per  cent  saline  solution. 

(c)  Now  place  it  in  the  iodine  solution  (iod.   1,  pot. 
iod.  2,  and  water  100)  for  two  to  three  minutes. 

(d)  Wash  it  again  in  '6  per  cent  saline  solution. 

(e)  Decolourise  it  with  the  aniline  oil  and  xylol  mixture 
(vide  p.  52). 

(/)  Wash  thoroughly  with  xylol  to  remove  all  the 
aniline  oil. 

(g) '  Mount  and  examine  it. 

In  most  cases  the  fibrin  network  of  the  diphtheritic 
membrane  will  be  beautifully  stained,  and  the 
bacilli  always  stand  out  well. 

It  is  desirable  when  dealing  with  paraffin  sections, 
fixed  on  cover -glasses,  to  perform  the  various  washings, 
decolourisations,  counterstainings,  and  clearings  in  wide- 
necked  bottles,  holding  the  cover-glasses  in  a  pair  of 
forceps. 


LESSON  XV 

Tubercle  Bacillus  (concluded] — Films  of  Pure  Cultures — Avian  Tuber- 
culosis —  Glanders  —  Tetanus  Bacillus  —  Paraffin  Sections  of  Ac- 
tinomycosis  —  Pyaemia  —  Phagocytosis  in  the  Frog  —  Inoculation 
of  the  Pithed  Frog — Hanging  Drops  of  Frog's  Lymph — Effect  of 
Temperature  on  Phagocytosis. 


Tubercle  Bacilli  (pure  cultures) 

(1)  Mammalian  Tubercle. 

(a)  Prepare  cover-glass  films  from  the  emulsion  supplied. 

(b)  Stain    them  with  warm  carbol   fuchsine  or  carbol 
gentian- violet,  decolourise  with  25  per  cent  hydrochloric 
acid  and  70  per  cent  alcohol,  as  in  the  case  of  tubercular 
sputum.     Counterstaining  is,  of  course,  unnecessary,  since 
no  other  organisms  are  present. 

(2)  Avian  Tubercle. 

Prepare  cover-glass  films  from   the  emulsion  supplied, 

and  proceed  as  above. 

• 

Examine  with  ^  in.  oil  immersion  and  compare  the 
two  kinds  of  bacilli. 


GENERAL  BACTERIOLOGY  LESSON 


Glanders— Horse's  Lung  (paraffin  sections) 

(i.)  Slow  Method. 

(a)  Fix  the  sections  on  clean  cover-glasses  and  dissolve 
out  the  paraffin  (vide  p.  64). 

(b)  Place  them  in  Loffler's  methylene-blue  for  six  to  eight 
hours. 

(c)  Wash  them  in  distilled  water  or  in  '6  per  cent  saline 
solution. 

(d)  Then  place  them  in  a  solution  of  tannic  acid  (1  in 
10)  for  four  to  five  hours. 

(e)  Wash  thoroughly  in  water. 
(/)  Dehydrate  in  absolute  alcohol 
(g)  Clear  in  xylol  and  mount. 

Examine  with  a  high  power  and  ^  in-  °il  immersion, 
(ii.)  Quick  Method. 

(a)  Fix  the  sections  on  clean  cover-glasses  and  dissolve 
out  the  paraffin  as  before. 

(b)  Float  the  sections  for  ten  to  thirty  seconds   on  a 
solution  of  carbol  methylene-blue  (i.e.  a  saturated  solution 
of  methylene-blue  in  5  per  cent  carbolic  acid,  which  must 
always  be  filtered  before  use). 

(c)  Then  wash  the  sections  in  water. 

(d)  Wash  them  in  a  10  per  cent  solution  of  tannic  acid 
for  half  a  minute  to  one  minute. 

(e)  Counterstain  in  a  weak  solution  of  cosine  or  acid 
fuchsine  in  10  per  cent  tannic  acid,  till  the  sections  are  red. 


STAINING  OF  TETANUS  BACILLI 


(/)  Now  wash  them  in  water  till  they  are  pink. 

(g)  Dehydrate  in  absolute  alcohol,  clear  in  xylol  and 
mount. 

Examine  with  a  high  power  and  -J^  in.  oil  immersion. 
The  glanders  bacilli  are  blue  on  a  pink  back- 
ground. 

Tetanus 
Broth  Culture. 

(a)  Prepare  cover-glass  films  in  the  usual  manner,  and 
pass  them  three  times  through  the  flame. 

(6)  Clear  them  in  acetic  acid,  wash  and  dry,  and  pass 
them  again  through  the  flame. 

(1)  Stain  some  of  the  films  with  Loffler's  methylene-blue 
or  gentian- violet  for  two  to  three  minutes. 

Examine  with  -^  in.  oil  immersion.  Observe  the 
drum-stick  shape  of  the  spore-bearing  bacilli. 

(2)  Stain  other  cover-glass  films  for  spores  with  Loffler's 
fuchsine  and  methylene-blue  (vide  p.  31). 

Examine  with  -^  in.  oil  immersion.     The  terminal 
spores  are  stained  red. 

Paraffin  Sections  of  Actinomycosis  Hominis 

(1)  Weigerf s  fibrin  method. 

(a)  Fix  the  sections  on  clean  cover-glasses  and  dissolve 
out  the  paraffin  (vide  p.  64). 

(&)  Stain  with  picrocarmine  for  half  an  hour. 


70  GENERAL  BACTERIOLOGY  LESSON 

(c)  Wash  in  water,  and  then  place  in  alcohol  for  half  a 
minute. 

(d)  Now  float  the  cover-glass  on  aniline  gentian-violet 
for  five  to  fifteen  minutes,  and  proceed  as  described  above. 

Examine  with  high  and  low  powers  and  -^  in.  oil 
immersion.  The  central  mycelium  is  stained  blue, 
while  the  clubs,  if  present,  generally  remain 
unstained  or  are  light  yellow. 

(2)  Differential  staining  of  dubs  and  mycelium. 

(a)  Fix  the  sections  on  clean  cover-glasses  and  dissolve 
out  the  paraffin. 

(b)  Stain  in  a  2  per  cent  aqueous  solution  of  rubine  for 
two  hours. 

(c)  Wash  in  water  till  the  sections  are  dark  red. 

(d)  Pass  quickly  through  spirit,  and  then  again  wash  in 
water  till  the  sections  are  pink. 

(e)  Stain  in  Loffler's  methylene-blue  for  half  a  minute 
to  one  minute. 

(/)  Wash  in  alcohol  till  the  sections  are  almost  pink 
again. 

(g)  Clear  in  xylol  and  mount. 

Examine  with  a  high  power  or  -^  in.  oil  immersion. 
The  clubs  are  red  and  the  mycelium  blue. 


Paraffin  Sections  of  Pysemic  Spleen  (Streptococcus) 
stained  by  Gram's  Method 

(a)  Fix  and  prepare  the  sections  as  before. 


xv  PHAGOCYTOSIS  IN  THE  FROG  71 

(b)  Stain  in  aniline  gentian -violet  for  half  an  hour. 

(c)  Wash  in  '6  per  cent  saline  solution: 

(d)  Place  in  Gram's  iodine  solution  for  half  a  minute. 

(e)  Again  wash  in  '6  per  cent  saline  solution. 

(/)  Now  wash  in  absolute  alcohol  for  half  a  minute. 
(g)  Quickly  pass  through  3  per  cent  hydrochloric  acid 
alcohol  (five  to  ten  seconds). 

(h)  Again  wash  in  absolute  alcohol  (ten  seconds). 

(i)  Stain  with  cosine  (eosine  -5  gramme,  50  per  cent 
alcohol  100  cc.)  for  a  second  or  two. 

(Jc)  Wash  in  absolute  alcohol,  till  the  section  is  pink  and 
apparently  quite  free  from  gentian-violet. 

(I)  Clear  in  xylol  and  mount. 

Here  again,  as  previously  mentioned,  the  various 
solutions  for  washing,  etc.,  are  best  kept  in  wide-necked 
bottles  or  pots. 

Phagocytosis 

A  pithed  frog  has  been  inoculated  under  the  skin  of  the 
thigh,  or  into  the  peritoneal  cavity,  with  a  few  drops  of  a 
young  broth  culture  of  anthrax  bacilli. 

Four  to  six  hours  later  examine  its  lymph  or  peritoneal 
fluid. 

(1)  Examination  in  a  hanging  drop. 

(a)  Pass  a  fine  capillary  tube  under  the  skin  of  the 
thigh,  or  into  the  peritoneal  cavity,  and  remove  some  of  the 
fluid,  and  carefully,  avoiding  air-bubbles,  blow  a  drop  on  to 


72  GENERAL  BACTERIOLOGY  LESSON 

the  centre  of  a  clean  cover-glass,  sterilised  by  passing  it 
several  times  through  the  flame. 

(b)  Place  the  cover-glass,  with  the  drop  downwards,  on  a 
moistened  ring  of  blotting-paper  (vide  Fig.  1). 

Examine  the  drop  with  a  high  power  and  with  y^  in. 
oil  immersion  for  phagocytosis  (vide  footnote  p.  10). 

(2)  Examination  of  stained  specimens. 

(a)  Spread  some  of  the  lymph  or  peritoneal  fluid  uni- 


FlG.  1. 

a,  Cover-glass  ;  5,  moistened  ring  of  blotting-paper  ;  c,  slide ;  d,  drop 
of  lymph  or  peritoneal  fluid. 

formly  over  several  cover-glasses,  and  allow  the  films  to  dry 
in  the  air. 

(b)  Clear  in  acetic  acid  in  the  usual  manner. 

(c)  Wash  in  water,  dry,  and  pass  three  times  through  the 
flame. 

(d)  Stain  with  Loffler's  methylene-blue,  wash,  dry  and 
mount. 

Examine  with  ^  in>  °^  immersion  for  phagocytosis. 
(3)  Examination  of  double-stained  specimens. 

(a)  Prepare  films  as  above. 

(b)  Stain  with  a  solution  of  cosine  (-5  gramme  in  100  cc. 
of  50  per  cent  alcohol)  for  half  a  minute,  without  previously 
clearing  in  acetic  acid. 


xv          PHAGOCYTOSIS  AND  GENERAL  INFECTION  73 

(c)  Wash   in   distilled   water,  and   dry  between  filter- 
paper. 

(d)  Pass  four  to  six  times  through  the  flame. 

(e)  Stain   in   Loffler's    methylene  -  blue   for  about  ten 
seconds. 

(/)  Wash  again,  dry  and  mount. 

Examine  with  a  high  power  and  ^  i*1-  °^  immersion 
for  phagocytes  and  eosinophile  cells.  Observe  the 
relation  of  the  latter  to  the  bacilli. 

(4)  Local  immunity  and  general  infection. 

(a)  Inoculate  a  pithed  frog  under  the  skin  of  the  thigh 
with  a  few  drops  of  a  fresh  broth   culture   of  Bacillus 
anthracis. 

(b)  To  do  this,  with  a  fine-pointed  pair  of  scissors  snip 
a  small  hole  in  the  skin  of  the  ventral  surface  of  the  thigh, 
just  above  the  knee. 

(c)  With  a  fine  capillary  tube  blow  a  small  quantity  of 
the  broth  culture  under  the  skin. 

(d)  Half  an  hour  later,  open  the  chest  of  the  frog  and 
expose  the  heart. 

(e)  With  a  sterile  capillary  tube  remove  some  of  the 
heart's  blood,  blow  it  over  the  surface  of  a  sloped  gelatine 
tube,  and  then  distribute  the  blood  uniformly  by  means  of 
a  platinum  needle. 

(/)  Place  the  tube  in  the  incubator  at  22°  C.  Twenty- 
four  or  forty-eight  hours  later  examine  it  for  colonies  of 
Bacillus  anthracis  and  make  impression  specimens  (vide 
p.  26). 


74  GENERAL  BACTERIOLOGY 

Examine  with  low  and  high  powers.     It  will  be  seen 
that  the  anthrax  bacilli  had  entered  the  circulation. 

(5)  Effect  of  heat  on  phagocytosis  in  the  frog. 

(a)  Inoculate  a  pithed1  frog  with  a  broth  culture  of 
Bacillus  anthracis  as  before,  and  keep  it  in. a  moist  chamber 
at  30°  to  35°  C.,  covering  it  over  with  moistened  blotting- 
paper. 

(b)  Examine  the  lymph  six  to  eighteen  hours  later,  by 
means  of  cover-glass  films,  stained  with  methylene-blue  in 
the  usual  manner. 

It  will  be  seen  that  the  bacilli  have  grown  luxuriantly, 
and  that  there  is  no  phagocytosis. 

Other  films  should   be   prepared  and  stained  with 
cosine  and  methylene-blue. 

It  will  be  seen  that  there  are  very  few  eosinophile 
cells. 

1  In  this  experiment  the  cord  alone  should  be  destroyed. 


LESSON  XVI 

Phagocytosis  (concluded)— Effect  of  Anaesthesia — Phagocytosis  in  the 
Hanging  Drop — Chemiotaxis — Diagnosis  of  Diphtheria. 

Phagocytosis  (concluded} 
(1)  Heated  frog. 

(a)  Remove,  with  a  capillary  pipette,  some  lymph  from 
the  inoculated  thigh  of  a  frog  kept  at  30°  to  35°  C.  for 
twelve  to  eighteen  hours  (vide  p.  74). 

Examine  it  as  a  hanging  drop  with  a  high  power, 
(vide  p.  72). 

Note  that  the  anthrax  bacilli  have  grown  luxuriantly, 
and  that  there  is  no  phagocytosis  or  "  eosinophile 
leucocytosis." 

(b)  Prepare  films  of  this  lymph,  dry  and  stain  them 

(1)  with  Loffler's  methylene-blue ; 

(2)  with  eosine  and  methylene-blue. 

Examine  them  with  a  high  power  and  ^  in.  oil 
immersion. 

(c)  Now  take  the  frog  out  of  the  incubator  and  keep  it 
cool  for  twenty-four  to  forty-eight  hours. 


76  GENERAL  BACTERIOLOGY  LESSON 

Then  examine  its  lymph  again  by  means  of  hanging 
drops  and  stained  films. 

Note  the  disappearance  of  the  anthrax  bacilli,  the 
well-marked  phagocytosis  and  "eosinophile  leuco- 
cytosis." 

(2)  Ancesthetised  frog. 

A  frog  has  been  anaesthetised  (chloroform-ether  mixture), 
and  during  the  narcosis  inoculated  under  the  skin  of  the 
right  thigh  with  a  virulent  culture  of  anthrax  bacilli ;  and 
it  has  then  been  kept  at  the  ordinary  temperature  in  a  moist 
chamber. 

Twenty-four  hours  later  it  has  been  pithed. 

Now  prepare  hanging  drops  and  stained  films  of  its 
lymph. 

Examine  them  with  a  high  power  and  -^  in-  oil 
immersion. 

Note  that  the  anthrax  bacilli  have  grown  well,  and 
that  there  is  no  phagocytosis  or  "eosinophile 
leucocytosis." 

This  proves  that  the  immunity  of  the  frog  from  anthrax 
does  not  depend  merely  on  the  body  temperature. 

(3)  Phagocytosis  in  the  hanging  drop. 

(a)  Prepare  a  moist  chamber,  as  described  on  p.  15. 

(b)  Pith  a  healthy  frog,  and  withdraw  some  of  its  lymph 
or  peritoneal  fluid  in  the  usual  manner. 

(c)  Place  a  drop  of  the  lymph  or  fluid  on  the  centre  of  a 
sterile  cover-glass,  avoiding  air-bubbles. 


xvi  CHEMIOTAXIS  77 

(d)  Carefully  inoculate  this  drop  from  a  fresh  broth 
culture  of  Bacillus  anthracis,  using  a  small  platinum  loop 
and  avoiding  over-inoculation. 

(e)  Place  the  cover-glass,  drop  downwards,  on  the  moist 
ring  of  filter-paper. 

Examine  with  a  high  power,  or  ^  in.  oil  immersion, 
using  a  narrow  diaphragm. 

Choose  a  good  field  for  observation,  one  containing  a 
short  chain  of  bacilli  and  numerous  cells. 

Note  that  the  coarsely  granular  cells  attack  the  bacilli 
first,  and  that  the  phagocytes  become  active  later. 

Eventually  take  off  the  cover-glass,  carefully  allow 
the  film  to  dry,  and  stain  it  with  cosine  and 
methylene-blue  in  the  usual  manner. 

Examine  it  with  ^  in.  oil  immersion,  and  note 
especially  the  relation  of  the  eosinophile  cells  and 
phagocytes  to  the  bacilli. 

Chemiotaxis 

(a)  Inject  a  small  quantity  of  an  old  broth  culture  of 
Bacillus  pyocyaneus  under  the  skin  of  the  thigh  of  a  pithed 
frog. 

(&)  Four  hours  later  remove  some  of  the  lymph  and 
prepare  films. 

(c)  Stain  these  in  the  usual  manner  with  cosine  and 
methylene-blue. 

Examine  with  a  high  power  and  -^  in.  oil  immersion. 
Note  the  large  number  of  eosinophile  cells  attracted 
("  positive  chemiotaxis  "). 


78  DIAGNOSIS  OF  DIPHTHERIA  LESSON  xvi 


Examination  of  Diphtheritic  Membrane 

(a)  Wash  the  membrane  with  sterilised  saline  solution 
in  a  sterile  watch-glass. 

(b)  Snip   off   a   small  piece   with   a   sterilised   pair  of 
scissors,  place  it  in  another  watch-glass  containing  saline 
solution,  and  with  a  sterile  glass  rod  break  it  up. 

(c)  Dip  a  small  platinum  loop  into  the  milky  suspension 
and  inoculate  three  serum  agar-agar  tubes  in  the  following 
manner  : — 

Make  three  parallel  streaks  on  the  surface  of  the 
first  tube ;  without  recharging  the  loop  make  three 
similar  streaks  on  the  surfaces  of  the  second  and 
third  tubes. 

(d)  Place  the  tubes  in  the  warm  incubator, 
Examine  next  morning  as  follows  : 

(1)  Place  a  small  drop  of  sterile  water  on  a  clean  cover- 
glass,   select   a   suspicious   colony,   and   with   a   platinum 
needle  transfer  it  to  the  cover-glass.     Gently  press  the  latter 
on  to  a  slide  and  examine  unstained. 

(2)  Proceed  in  the  same  manner  but  allow  the  drop  to 
dry.      Pass   the   film   through  the  flame  and   stain  with 
gentian- violet  (vide  p.  57). 

The  diphtheria  bacilli  are  easily  recognised  by  their 
form  and  grouping,  and  by  the  absence  of  motility. 


PAKT    II 
BACTERIOLOGICAL   ANALYSIS 

LESSONS    I-XII 


LESSONS  I  AND  II 

Cleaning  and  Sterilisation  of  Tubes  and  Flasks  —  Preparation  of 
Nutrient  Media  —  Beef  Broth  —  Glycerine  Broth  —  Grape-Sugar 
Broth — Meat  Infusion — Gelatine — Grape-Sugar  Gelatine — 25  per 
cent  Gelatine — Carbolic  Acid  Gelatine — Peptone  Solution— Potato 
Tubes — Milk  Tubes — Agar-Agar— Glycerine  Agar-Agar— Grape- 
Sugar  Agar-Agar— Serum  Tubes. 

Cleaning  and  Sterilisation  of  Test-Tubes, 
Flasks,  and  Beakers 

I.  CLEANING  OF  NEW  TEST-TUBES. 

(1)  Eoll  a  little  cotton -wool  round  a  strong  glass  rod 
and  fasten  it  with  a  piece  of  strong  thread  or  string. 

(2)  Dip   the   cotton -wool   in   strong   nitric   acid,    and 
thoroughly  rub  the  inside  of  each  tube. 

(3)  Wash  the  tubes  three  or  four  times  in  water,  to 
remove  all  trace  of  the  acid. 

(4)  Allow  them  to  drain,  until  they  are  nearly  dry. 

(5)  Rinse  each  tube  with  a  little  absolute  alcohol  or 
strong  spirit. 

(6)  Again  allow  the  tubes  to  drain. 

(7)  When  quite  dry,  they  are  ready  for  sterilisation. 


82  BACTERIOLOGICAL  ANALYSIS  LESSONS 

II.  CLEANING  OF  TEST-TUBES  WHICH  HAVE  ALREADY 

BEEN   USED. 

(1)  Remove  the  cotton-wool  plugs. 

(2)  Steam  the  tubes  in  the  steriliser  or  autoclave  for 
thirty  minutes,  in  order  to  dissolve  the  gelatine  or  agar- 
agar,  and  to  disinfect  the  tubes. 

(3)  Pour  away  the  contents  of  the  tubes,  and  place  the 
empty  tubes,  upright,  in  a  large  sauce-pan  containing  a  weak 
solution  of  caustic  soda. 

(4)  Fill  each  tube  with  the  same  caustic  soda  solution, 
boil  for  an  hour,  and  then  allow  the  sauce-pan  to  cool. 

(5)  Pour  away  the  caustic  soda  solution,  and  wash  the 
tubes  three  or  four  times  in  water. 

(6)  Now  clean  each  tube  with  weak  nitric  acid,  using  a 
glass  rod  to  which  a  little  cotton-wool  has  been  fastened. 

(7)  Wash  the  tubes  repeatedly  in  water. 

(8)  Einse  them  with  a  little  absolute  alcohol,  and  allow 
them  to  drain. 

(9)  When  dry,  they  are  ready  for  sterilisation. 

Very  old  tubes  in  which  the  culture  media  have  dried 
up  must  be  discarded,  since  no  amount  of  cleaning 
will  make  them  fit  for  use. 

III.  CLEANING  OF  FLASKS  AND  BEAKERS. 

(1)  Immediately  after  use  rinse  out  the  flask  or  beaker 
with  hot  water. 

(2)  With  a  piece  of  bent  wire,  to  which  a  little  cotton- 
wool is  tied,  and  which  is  charged  with  sapolio,  clean  the 


r  AND  ii  CULTURE  MEDIA  83 

inside  of  the  vessel  until  all  dirt,  stains,  albumen,  and  fat 
are  removed. 

(3)  Wash  thoroughly  in  hot  water,  and  then  with  nitric 
acid.     If  the  flasks  or  beakers  are  much  stained,  allow  the 
acid  to  act  for  some  little  time. 

(4)  Rinse  thoroughly  with  tap  water  and  finally  with 
distilled  water. 

(5)  Allow  the  vessels  to  drain ;  and  when  dry,  they  are 
ready  for  sterilisation. 

IV.  STERILISATION  OF  TEST -TUBES,  FLASKS,  AND 
BEAKERS. 

Place  the  tubes  and  vessels  when  clean  in  the  hot-air 
steriliser  for  half  an  hour  to  three  quarters  of  an  hour, 
having  previously  plugged  them  with  cotton-wool. 

Sterilisation  is  completed  when  the  cotton-wool  plugs 
turn  slightly  brown. 


Preparation  of  Nutrient  Media 
I.  PREPARATION  OF  BEEF  BROTH. 

(1)  Take  one  pound  of  lean  beef,  remove  all  fat  and 
connective  tissue,  and  cut  it  up  into  small  pieces. 

(2)  Mince  it  in  a  sausage  machine  or  chop  it  up  finely. 

(3)  Add  1000  cc.  of  distilled  water  and  stir  vigorously. 
Then  boil  for  half  an  hour  to  one  hour  in  a  large  flask  or 
in  an  enamelled  iron  pan,  which  must  be  well  covered  up. 

(4)  Filter,  and  make  the  filtrate  up  to  1000  cc.  with 

distilled  water. 


84  BACTERIOLOGICAL  ANALYSIS  LESSONS 

(5)  Pour  it  into  a  large  flask  containing  5  grammes  of 
sodium  chloride  and  10  grammes  of  pure  peptone. 

(6)  Heat  this  mixture  in  a  water  bath  or  in  a  steam 
steriliser,  at  100°  C.,  for  an  hour,  shaking  the  flask  from 
time  to  time,  so  as  to  prevent  the  peptone  from  being 
charred. 

(7)  Now  neutralise  carefully  with  a  concentrated  solution 
of  sodium  carbonate,  making  the  solution  faintly  alkaline. 

(Should  the  solution  have  been  made  too  alkaline, 
add  a  little  lactate  of  ammonium,  till  the  proper 
degree  of  alkalinity  is  reached.) 

(8)  After  neutralisation,  heat  for  another  half -hour  in 
the  water  bath  or  steam  steriliser,  shaking  the  flask  from 
time  to  time  as  before. 

(9)  Once  more  test  the  reaction,  and  if  unchanged,  i.e.  if 
slightly  alkaline,  filter  into  a  sterilised  flask  through  two 
layers  of  Swedish  filter-paper. 

(10)  Sterilise  on  two  successive  days  in  the  steamer, 
for  twenty  minutes  on  each  occasion.1 

To  fill  tubes  with  broth : — 

Take  a  number  of  cleaned  and  sterilised  test-tubes. 

1  If  after  the  first  sterilisation  a  precipitate  appears,  or  the  broth 
becomes  turbid,  allow  it  to  settle ;  then  filter  again  and  recommence 
the  sterilisation.  If,  in  spite  of  a  second  filtration,  the  broth  does 
not  remain  clear,  it  is  advisable  to  add  the  white  of  an  egg  to  the 
broth,  while  cool,  and  to  heat  it  gradually,  shaking  it  from  time  to 
time.  Heat  till  all  the  albumen  is  precipitated  and  carried  down  with 
the  white  of  egg.  Then  filter  again,  and  sterilise  on  three  successive 
occasions. 


I  AND  ii  MODIFICATIONS  OF  BEEF  BROTH  85 

Pour  some  broth  into  a  sterile  separating  funnel,  and 
allow  5  to  8  cc.  to  run  into  each  tube. 

Sterilise  these  broth-tubes  in  the  steamer,  for  twenty 
minutes,  on  three  successive  days. 

It  is  advisable  during  sterilisation  to  cover  up  the  tubes 
with  a  piece  of  stout  tinfoil,  so  as  to  prevent  the  steam 
from  condensing  on  the  cotton-wool  plugs. 

In  any  case,  when  nutrient  media  are  to  be  sterilised 
in  the  steamer,  care  should  be  taken  that  the 
latter  is  heated  up  to  100°  C.  before  the  tubes  or 
flasks  are  placed  in  it. 

Occasionally  after  sterilisation  a  slight  precipitate 
appears  in  the  broth.  This  will  settle  gradually, 
and  for  ordinary  purposes  may  be  disregarded. 

II.  GLYCERINE  BROTH  AND  GRAPE-SUGAR  BROTH. 

(a)  Glycerine  broth. 

To  every  100  cc.  of  broth  add  4  to  6  cc.  of  pure  glycerine, 
and  shake  the  flask  till  they  are  thoroughly  mixed. 

Fill  sterile  test-tubes,  and  sterilise  them  in  the  usual 
manner. 

(b)  Grape-sugar  Iroth. 

To  every  100  cc.  of  broth  add  2  grammes  of  grape-sugar. 

When  the  sugar  is  quite  dissolved,  fill  a  number  of 
sterile  test-tubes,  and  sterilise  them  in  the  usual  manner. 

Glycerine  broth  is  used  for  the  cultivation  of  tubercle 
bacilli,  and  grape-sugar  broth  for  anaerobic  cultiva- 
tion. 


86  BACTERIOLOGICAL  ANALYSIS  LESSONS 

III.  MEAT  INFUSION. 

(1)  Prepare  the  meat  as  before  (vide  p.  83). 

(2)  To  one  pound  of  the  minced  beef  add  1000  cc.  of 
distilled  water  and  allow  the  mixture  to  stand  in  a  cool 
place  for  twenty-four  hours,  having  previously  stirred  it 
vigorously. 

(3)  Next  day  strain  through  muslin,  and  press  as  much 
juice  as  possible  out  of  the  meat. 

(4)  Make  up  to  1000  cc.  with  distilled  water. 

(5)  Pour  the  strained  infusion  into  a  flask,  containing 
5  grammes  of  sodium  chloride  and  10  grammes  of  peptone, 
and  heat  in  a  water  bath  or  in  the  steamer  for  an  hour ; 
then  proceed  exactly  as  before  (vide  supra). 

(Koch  advised  the  preparation  of  bouillon  with  meat 
infusion.  This  method,  however,  has  no  special 
advantages  and  is  less  convenient,  requiring  more 
time.) 

IV.  GELATINE. 

(1)  Prepare  1000  cc.  of  broth  (vide  supra)  and  pour  it 
into  a  large  beaker,  containing  100  to  120  grammes  of  sheet 
gelatine  rolled  up. 

(2)  Allow  the  gelatine  to  soak  for  half  an  hour  to  one 
hour. 

(3)  Place  the  beaker  in  a  water  bath,  covering  it  up 
with  a  clean  glass  plate,  and  heat  the  bath  slowly  and 
steadily,  without  letting  it  reach  boiling-point. 

(4)  When  the  gelatine  has  been  dissolved  neutralise 


I  AND  ii  GELATINE  TUBES  87 

carefully,  and  heat  for  another  half -hour,  stirring  it  from 
time  to  time. 

(5)  Then  add  the  white  of  an  egg,  and  continue  heating 
till  all  the  albumen  is  precipitated. 

(6)  Filter  through  a  hot- water  funnel  with  two  layers  of 
moistened  niter-paper  into  sterilised  flasks,  and  sterilise  in 
the  steamer  on  two  successive  days  for  twenty  minutes.1 

To  fill  tubes  with  gelatine : — 

Melt  the  gelatine  and  pour  it  inta  a  sterile  separating 
funnel,  and  proceed  as  described  for  broth-tubes  (vide  p.  84). 

The  gelatine  tubes  must  be  sterilised  in  the  steamer  on 
two  successive  days. 

After  the  last  sterilisation,  place  some  of  the  tubes, 
while  still  liquid,  on  a  sloped  tray  and  allow  the  gelatine 
to  set  in  this  manner  (for  streak  cultures).  Allow  the  others 
to  set  in  an  upright  position  (for  stab  cultures  and  plates). 

V.  GRAPE-SUGAR  GELATINE. 

(1)  In  every  100  cc.  of  liquid  gelatine  dissolve  2  grammes 
of  grape-sugar. 

(2)  Fill  test-tubes,  and  sterilise  on  two  successive  days 
in  the  usual  manner. 

VI.  25  PER  CENT  GELATINE. 

(1)  To  every  100  cc.  of  liquid  10  per  cent  gelatine  add 

1  Gelatine  must  not  be  heated  too  much  nor  too  long,  since  by  so 
doing  it  may  lose  its  property  of  setting  when  cold.  The  gelatine 
should  be  quite  clear.  If  it  be  turbid  after  sterilisation,  this  is  often 
due  to  the  fact  that  it  is  too  alkaline.  In  such  a  case  add  lactate  of 
ammonium,  not  hydrochloric  or  nitric  acid. 


88  BACTERIOLOGICAL  ANALYSIS  LESSONS 

15  grammes  of  sheet  gelatine,  and  heat  slowly  in  a  beaker, 
placed  in  a  water  bath,  till  all  the  gelatine  is  dissolved. 

(2)  Neutralise  in  the  usual  manner,  and  again  heat  for 
half  an  hour  to  one  hour. 

(3)  If  necessary,  clear  with  the  white  of  an  egg. 

(4)  Then  filter  through  a  hot- water  funnel  into  a  sterilised 
flask. 

(5)  Fill  test-tubes  in  the  usual  manner,  and  sterilise  them 
on  two  successive  days. 

Grape-sugar  gelatine  is  employed  for  anaerobic 
cultivation,  and  25  per  cent  gelatine  is  especially 
useful  in  the  diagnosis  between  the  bacillus  of 
typhoid  fever  and  the  Bacterium  coli  commune. 

VII.  CARBOLIC  ACID  GELATINE. 

(1)  To  every  100  cc.  of  liquid  10  per  cent  gelatine  add 
4  cc.  of  a  5  per  cent  solution  of  pure  carbolic  acid. 

(2)  Fill  test-tubes  in  the  usual  manner,  and  sterilise  them 
on  two  successive  days. 

This  gelatine  is  most  useful  for  the  separation  of 
typhoid  bacilli  or  of  the  Bacterium  coli  commune. 

VIII.  PEPTONE  SOLUTION. 

(1)  To  10  grammes  of  pure  peptone  and  5  grammes  of 
sodium  chloride  add  1000  cc.  of  distilled  water. 

(2)  Boil  for  an  hour,  neutralise  carefully  in  the  usual 
manner,  and  boil  for  another  half-hour. 

(3)  Filter  into  sterilised  flasks,  and  sterilise  in  the  auto- 


I  AND  ii  MILK  AND  POTATO  TUBES  89 


clave  at  120°  C.  for  twenty  minutes:  a  single  sterilisation 
suffices,  but  overheating  must  be  carefully  avoided. 

(4)  Fill  test-tubes  in  the  usual  manner,  and  sterilise  them 
in  the  autoclave. 

IX.  POTATO  TUBES. 

(1)  Take  sound  large  potatoes  and  scrub  them  thoroughly. 

(2)  Cut  off  the  ends,  and  with  a  cork-borer  bore  solid 
cylinders  out  of  the  potatoes. 

(3)  These  cylinders  should  measure  3|-  to  4  in.  in  length, 
and  just  fit  the  lumen  of  the  test-tubes. 

(4)  By  means  of  a  diagonal  cut  divide  them  in  two, 
and  wash  them  thoroughly  in  water,  which  may  be  slightly 
alkaline. 

(5)  Now  take  sterilised  test-tubes,  and  at  the  bottom 
of  each  place  a  little  moistened  cotton-wool.      Into  each 
tube  drop  a  piece  of  potato,  with  the  broad  end  downwards, 
and  replace  the  cotton-wool  plugs. 

(6)  Sterilise  in  the  autoclave  for  half  an  hour  to  one 
hour  at  120°  C.,  covering  the  tubes  up  with  stout  tinfoil. 
Avoid  overheating,  otherwise  the  potatoes  will  lose  their 
natural  white  colour  and  become  sodden  in  appearance. 

X.  MILK  TUBES. 

(1)  Neutralise  "  separated  milk,"  if  it  be  acid,  and  fill  a 
number  of  sterile  test-tubes,  in  the  ordinary  manner,  using 
a  separating  funnel. 

(2)  Sterilise  on  three  successive  days  in  the  steamer. 


90  BACTERIOLOGICAL  ANALYSIS  LESSONS 

XI.  AGAR-AGAR. 

(1)  Weigh  out  10  grammes  of  agar-agar  fibre,  cut  it  up 
finely,  and  allow  it  to  swell  in  a  very  dilute  solution  of 
acetic  acid  (3  to  4  cc.  of  glacial  acetic  acid  to  500  cc.  of 
water)  tor  fifteen  minutes. 

(2)  Drain  away  the  acetic  acid. 

(3)  Wash  the  agar-agar  in  distilled  water,  to  remove  all 
trace  of  acid. 

(4)  Add  the  washed  agar-agar  to  500  cc.  of  broth  and 
boil :  it  will  dissolve  in  about  fifteen  to  thirty  minutes. 

(5)  Neutralise  carefully — very  little  sodium  carbonate  is 
required — and  boil  again  for  a  few  minutes. 

(6)  Allow  it  to  cool  somewhat  •  then  clear  it  by  adding 
the  white  of  an  egg,  and  heating  it  in  the  autoclave  for  a 
half  to  three-quarters  of  an  hour  or  even  longer. 

(7)  Now  filter  through  a  hot-water  funnel. 

To  hasten  the  filtration,  if  necessary,  change  the  filter- 
paper  frequently,  and  always  keep  the  unfiltered  agar-agar 
hot  (see  note  p.  92). 

(8)  Sterilise  the  filtered  agar-agar  in  the  autoclave  for 
twenty  to  thirty  minutes  at  120°  C.  :  a  single  sterilisation 
is  sufficient. 

Agar-agar  prepared  in  this  way  will  often  filter  as 
quickly  as  gelatine,  and  is  exceedingly  clear. 

To  fill  tubes  with  agar-agar : — 

Melt  the  agar-agar  in  the  autoclave  and  pour  it  into  a 
sterile  separating  funnel,  and  fill  the  tubes  in  the  usual 
manner. 


i  AND  ii  AGAR-AGAR  TUBES 

The  tubes  afterwards  must  be  sterilised  in  the  autoclave, 
jing  at  the  same  time  covered  up  with  stout  tinfoil. 

When  the  sterilisation  is  complete,  place  some  tubes  on 
sloping  tray  and  allow  the  agar-agar  to  set  on  the  slant ; 
illow  the  other  tubes  to  set  in  an  upright  position. 

The  latter  may  be  sloped  at  any  time  by  again  melting 
le  agar-agar  in  the  autoclave  and  placing  the  tubes  on 
te  sloping  tray. 

XII.    GLYCERINE    AGAR-AGAR    AND    GRAPE-SUGAR 
AGAR-AGAR. 

(a)  Glycerine  agar-agar. 

(1)  To  every  100  cc.  of  liquid  agar-agar,  add  5  to  6  cc.  of 
pure  glycerine,  and  mix  thoroughly. 

(2)  Fill  test-tubes  in  the  usual  manner,  and  sterilise 
them  in  the  autoclave. 

(3)  Some  tubes  should  be  allowed  to  set  on  the  slant, 
others  in  an  upright  position. 

(b)  Grape-sugar  agar-agar. 

(1)  To  every  100  cc.  of  liquid  agar-agar  add  2  grammes  of 
grape-sugar,  and  shake  till  the  sugar  is  dissolved. 

(2)  Fill  test-tubes  in  the  usual  manner,   and  sterilise 
them  in  the  autoclave  at  120°  C. 

(3)  Allow  the  agar-agar  to  set  in  an  upright  position. 

If  the  grape  -  sugar  agar  -  agar  is  to  be  used  for 
anaerobic  cultivations,  the  tubes  must  be  filled  to 
two-thirds  of  their  height. 


92  BACTERIOLOGICAL  ANALYSIS        LESSONS  i,  n 

To  prevent  the  agar-agar  from  slipping,  after  having 
been  slanted,  allow  it  to  cool  and  set  slowly,  and 
keep  the  tubes  for  one  or  two  days  at  the  tempera- 
ture of  the  room.1 

XIII.  SERUM  AGAR-AGAR. 

(1)  Take  100  cc.  of  fresh  ascitic  or  pleuritic  fluid. 

(2)  Add  2  to  2-5  cc.  of  a  10  per  cent  solution  of  caustic 
soda,  shake  gently  and  heat  in  the  steamer  for  an  hour. 

(3)  Now   add    1*5    grammes   of  agar-agar,    treated   as 
described  on  p.  90. 

(4)  Heat  in  the  autoclave  for  about  two  hours,  filter  and 
fill  tubes  in  the  ordinary  manner. 

Serum  agar-agar  tubes  are  of  value  in  the  cultivation 
and  separation  of  diphtheria  bacilli,  for  diagnostic 
purposes.2 

1  In  preparing  agar-agar  according  to  the  above  method  it  is  not 
necessary  to  clear  with  the  white  of  an  egg,  though  this  considerably 
improves  the  clearness  of  the  medium.     To  hasten  nitration,  in  case 
this  should  be  slow, — as  occasionally  happens, — instead  of  changing  the 
papers  the  hot  agar-agar  may  be  first  passed  through  Papier  Chardin 
(Cogit  and  Co.,  Paris),  and  then  through  two  layers  of  Swedish  filter- 
paper.     In  any  case,  before  filtering,  the  agar-agar  should  be  heated  up 
properly. 

2  The  addition  of  5  per  cent  glycerine  and  1  per  cent  grape-sugar  is 
advisable. 


LESSON  III 

lination  of  Water — I.  Quantitative  Examination  by  Plate  Culture 
Method  and  Roll  Tubes— Effect  of  Sunlight  on  Water. 

I.  Quantitative  Examination  of  Water 

COLLECT  the  water  to  be  tested  in  a  sterilised  flask. 

(a)  If  tap  water,  allow  it  to  run  for  a  minute  or  two 
before  collecting  it. 

(&)  If  stagnant  water  or  river  water,  collect  it  from 
varying  depths  with  special  pipettes  or  bottles,  and  empty 
the  latter  into  sterilised  flasks. 

A.  TAP  WATER. 

(a)  Plate  culture  method. 

(1)  Collect  100  cc.  of  the  water  to  be  examined  in  a 
sterilised  flask,  and  shake  the  latter. 

(2)  With  a  sterile  graduated  pipette,  connected  with  a 
short  india-rubber  tube,  suck  up  *25  cc.  of  this  water,  and 
add  it  severally  to  each  of  three  liquefied  gelatine  tubes. 

To  sterilise  the  pipette,  it  should  be  plugged  with 
cotton-wool  at  its  broad  end  (a),  and  placed  in  a 
test-tube,  the  mouth  of  which  is  also  plugged  with 


BACTERIOLOGICAL  ANALYSIS 


LESSON 


cotton-wool  (b).    The  tube  and  pipette  should  then 
be  placed  in  the  hot-air  steriliser  (vide  Fig.  2). 
Before  using  the  pipette,  fix  a  thin  india-rubber  tube 
to  it,  without  removing  the  cotton-wool  plug. 

(3)  To  other  three  gelatine  tubes  add  severally  -1  cc.  of 
the  water. 


FIG.  2. 


FIG.  3. 


(4)  Gently  shake  the  gelatine  tubes,  and  prepare  plates 
in  the  usual  manner  (vide  p.  29). 

(5)  Keep  these  in  the  cool  incubator  at  22°  C. 

If  the  water  to  be  tested  be  suspected  of  containing 
large  numbers  of  bacilli,  smaller  quantities,  less 
than  '1  cc.,  should  be  added  to  the  gelatine.1 

1  The  pipettes  sold  by  Hawksley  are  well  adapted  for  measuring 
minute  quantities. 


in  COUNTING  COLONIES  IN  PLATES  95 

In  order  to'  keep  the  pipette  sterile,  after  it  has  been 
used,  push  it,  through  a  loosely  fitting  cotton-wool 
plug,  into  a  flask  containing  sterile  water,  which  is 
kept  at  boiling  point  over  a  gas  flame  (Fig.  3). 

Each  time  before  using  the  pipette,  of  course,  it  must 
be  allowed  to  cool. 

Examine  the  plates  from  day  to  day,  and  count  the 
colonies  which  appear,  in  the  following  manner  : — 

Cut  out  a  piece  of  paper,  fitting  exactly  the  under 
surface  of  the  Petri's  capsule,  and  divide  it  into 
sixteen  equal  segments,  of 
which  one  is  painted  black 
(vide  Fig.  4).  Fix  this  to 
the  under  surface  of  the 
capsule,  and  with  a  lens 
or  dissecting  microscope 
count  the  colonies  over 
the  black  segment.  Mul- 
tiply this  number  by  six- 
teen, and  the  result  will 

give  the  number  of  organisms  in  -25,   '1  cc.,  or 
whatever  quantity  of  water  was  used. 

(6)  Roll  tubes. 

(1)  Prepare  gelatine  tubes  as  before. 

(2)  Shake  them  gently,  so  as  to  distribute  the  water  and 
its  organisms  as  uniformly  as  possible. 

(3)  With  a  tube  containing  boiling  water  melt  a  groove 
in  a  block  of  ice. 


96  BACTERIOLOGICAL  ANALYSIS  LESSON 

(4)  Place  the  gelatine  tube,  containing  the  water  to  be 
tested,  in  this  groove,  keeping  it  horizontal;  and  roll  it 
rapidly  round  and  round,  until  the  gelatine  sets  on  the 
inner  surface  of  the  tube  in  a  uniform  layer. 

(5)  Place  these  roll  tubes  in  the  cool  incubator  at  22°  C. 

Examine  the  tubes  from  day  to  day,  and  count  the 
colonies,  before  liquefaction  sets  in. 

The  counting  must  be  done  by  means  of  Esmarch's 
apparatus. 

The  quantitative  examination  of  water  is  not  of  great 
value,  .except  for  the  purpose  of  testing  niters,  or  of 
comparing  different  kinds  of  water,  as  for  instance  tap 
water  and  distilled  water,  or  of  studying  the  effect  of 
physical  or  chemical  processes  on  a  given  sample  of 
water. 

B.  DISTILLED  WATER. 

Prepare  gelatine  tubes  and  work  them  up  in  exactly  the 
same  manner  as  described  above,  and  compare  the  result  of 
this  examination  with  that  of  the  previous  one. 

Fresh  distilled  water  contains  fewer  organisms  than  tap 
water. 

C.  TANK  WATER. 

Remove  two  samples  :  (a)  from  the  surface  j  (b)  from  a 
given  depth  (a  foot  below  the  surface). 

Work  these  samples  up  in  the  manner  already  described, 
using,  however,  not  more  than  -1  or  -12  cc. 

Compare  the  results :  the  surface  water  will  contain 
more  organisms  than  the  water  removed  from  the  depth. 


in  ACTION  OF  SUNLIGHT  ON  WATER  97 

D.  EFFECT  OF  SUNLIGHT  ON  WATER. 

(1)  Fill  a  tall  glass  cylinder  with  tank  water,  and  expose 
it  to  active  sunlight  for  several  hours,  having  first  removed 
a  sample  from  the  surface  by  means  of  a  sterile  pipette 
(Sample  A). 

(2)  Having  allowed  the  sun  to  act  on  the  water  for 
several  hours,  remove  a  second  sample  from  the  surface, 
and  another  from  the  depth  (Samples  B  and  C). 

Now  work  up  these  three  samples  in  the  usual  manner, 
using  not  more  than  1  to  '12  cc. 

Compare  the  results  : 

Sample  A  contains  most  organisms ; 
Sample  B  fewest. 

The  sun  has  little  or  no  action  on  the  Bacillus  fluor- 
escens  liquefaciens,  if  the  latter  should  be  present 
in  the  water. 


LESSON  IV 

Examination  of  Water  (continued} — II.  Qualitative  Examination- 
Examination  of  Water  for  the  Bacillus  of  Typhoid  Fever  and  the 
Bacterium  Coli  Commune — Indol  Reaction. 


II.  Qualitative  Examination  of  Water 

A.    EXAMINATION    OF  WATER    FOR    THE    BACILLUS    OF 
TYPHOID  FEVER  AND  THE  BACTERIUM  COLI  COMMUNE. 

Two  samples  of  water  are  supplied  (A)  and  (B),  of  which 
(A)  contains  typhoid  bacilli,  and  (B)  a  mixture  of  typhoid 
bacilli  and  the  Bacterium  coli  commune. 

(1)  Take  500  to  1000  cc.  of  each  sample  and  filter  through 
sterile  Berkefeld  filters  into  sterilised  flasks  (vide  Fig.  5), 
which  are  connected  with  an  air-pump. 

The  filters  should  be  previously  sterilised  in  the  auto- 
clave, the  flasks  in  the  hot-air  steriliser. 

(2)  Pour  10  cc.  of  the  filtered  water,  which  is  sterile, 
into   a   small   sterilised  beaker,  protected  with    a   sterile 
cotton-wool  plug. 

(3)  Now  aseptically  unscrew  the  "  candle  "  of  the  filter. 

(4)  With  a  soft  sterilised  tooth-brush  gently  scrape  the 


LESSON  iv      SEPARATION  OF  TYPHOID  BACILLUS  99 

u  candle  "  of  the  filter,  so  as  to  get  as  many  as  possible  of 
the  bacteria  from  the  surface  of  the  candle  into  the  beaker. 

(5)  The  water  in  the  beaker  will 
now  be  muddy  in  appearance  and 
contain  most  of  the  micro-organisms 
retained  by  the  filter. 

(6)  Plug  the  beaker  with  cotton- 
wool. 

(7)  Prepare    and    melt    several 
carbolic  acid  gelatine  tubes,  and  to 
each  add,  with  a  sterile  pipette,  "005 
to  '05  cc.  of  the  water  containing  the 
bacteria  scraped  off  the  filter. 

(8)  Pour    plates    in    the    usual 
manner,  and  place  them  in  the  cool 
incubator. 

The    growth   of   large   numbers 
of    micro-organisms,    especially   of 
the  liquefying  ones,  will  be  inhibited  by  the  carbolic  acid 
contained  in  the  gelatine. 

(9)  When  colonies  have  appeared,  make  subcultures  of 
those  in  any  way  resembling  the  bacillus  of  typhoid  fever 
or  the  Bacterium  coli  commune  : 

(a)  gelatine  streak  cultures ; 

(b)  gelatine  shake  cultures ; 

(c)  25  per  cent  gelatine  stab  cultures. 

Place  (a)  and  (b)  in  the  cool  incubator,  and  (c)  in  the 
warm  at  38 -5°  C. 


FIG.  5. 


100  BACTERIOLOGICAL  ANALYSIS        '    LESSON  iv 

(10)  Subsequently  select  those  cultures  which  apparently 
consist1  of  typhoid  bacilli  or  the  Bacterium  coli  commune, 
and  confirm  the  diagnosis  by 

(a)  microscopical  examination ; 
(6)  milk  cultures  (at  38-5°  C.) ; 
(c)  growth  on  potatoes  (at  38'5°  C.) 

For  differences  between  the  bacillus  of  typhoid  fever 
and  the  Bacterium  coli  commune  vide  p.  53. 

(11)  Test  also  for  the  presence  of  Indol. 

(a)  Prepare  broth  cultures  and  keep  them  at  38 '5  C. 
for  two  to  fourteen  days. 

(b)  Now  add  1  cc.  of  a  solution  of  potassium  nitrite 
(containing  -02  grammes  in  100  cc.),  and  then  a  few 
drops  of  pure  sulphuric  acid. 

If  Indol  is  present,  the  liquid  in  the  tube  turns  red. 

The  Bacterium  coli  commune  gives  a  positive  result, 
the  bacillus  of  typhoid  fever  a  negative  result. 


LESSON  V 

Examination  of  Water  (concluded]  —  Examination  of  Water  for  the 
Vibrio  Choleras  Asiaticse — Peptone  Method — Gruber's  Method— 
Agar-Agar  Plate  Method— Gelatine  Plate  Method. 

II.  Qualitative  Examination  of  Water  (concluded) 

B.  EXAMINATION  OF  WATER  FOR  THE  VIBRIO  CHOLERA 
ASIATICS. 

A  sample  of  water  is  supplied  containing  cholera  vibrios 
(500  cc.  of  distilled  water  containing  four  broth  tubes 
of  cholera  vibrios). 

I.  Peptone  method. 

(1)  To  separate  the  vibrios,   fill  four   small   sterilised 
flasks  with  50  cc.  of  2  per  cent  peptone  solution  each. 

(2)  To  these  add  respectively  50  cc.,  40  cc.,  25  cc.,  and 
10  cc.  of  the  suspected  water,  and  place  the  flasks  in  the 
warm  incubator  till  next  morning. 

(3)  Now  examine   the  surface  of  the  culture  fluid  for 
vibrios  by  means  of   cover- glass  films,  which  should  be 
stained  with  carbol  fuchsine  or  aniline  gentian-violet  (vide 
p.  40). 


102  BACTERIOLOGICAL  ANALYSIS  LESSON 

If  there  be  a  pellicle  on  the  surface  of  the  culture 
medium  in  these  flasks,  examine  it  for  vibrios. 

If  vibrios  are  present — 

(a)  Prepare  gelatine  plates  in  the  usual  manner,  keep 
them  at  22°  C.,  and  examine  them  from  day  to  day. 

Any  suspicious  colony  should  be  used  for  subcultiva- 
tion  : 

(1)  Under  a  dissecting  microscope  fish  out  the  colony 
with  a  thin  platinum  needle,  and  make  gelatine 
stab  cultures  and  agar-agar  streak  cultures. 

Keep  the  former  at  22°  C.  and  the  latter  at  38-5°  C. 

Examine  from  day  to  day,  and  prepare  cover-glass 
specimens,  which  should  be  stained  with  carbol 
fuchsine  or  aniline  gentian-violet  (vide  p.  40). 

(2)  From  the  suspicious  colonies  start  also  subcul- 
tures in  peptone  tubes. 

Place  them  in  the  warm  incubator  for  eighteen  to 
twenty-four  hours.  At  the  expiration  of  this  time 
examine  microscopically  for  vibrios. 

Apply  also  the  test  for  "  Cholera  Red  "  to  one  of  the 
tubes,  by  adding  a  few  drops  of  pure  concentrated 
•  sulphuric  acid. 

The  peptone  solution  should  acquire  a  markedly  red 
tint. 

(5)  Inoculate  also  a  series  of  peptone  tubes  from  the 
peptone  flasks,  and  keep  them  at  38*5°  C.  for  twenty-four 
hours. 

(1)  Then  test  one  of  these  tubes  for  "Cholera  Red." 


v  SEPARATION  OF  CHOLERA  VIBRIOS  103 

(2)  Examine  another  microscopically,  and  if  vibrios 
are  found  to  be  present,  prepare  gelatine  plates 
and  work  these  up  as  above. 

II.  Gruler's  method. 

(1)  Grow  cholera  vibrios  in  tubes,  containing  broth  or 
peptone  solution,  for  twenty-four  hours  at  38 '5°  C. 

(2)  Then  sterilise  these  tubes  by  heating  them  from 
60°  0.  to  65°  C.  for  ten  minutes  in  a  water  bath. 

Such  tubes  should  be  kept  in  readiness. 

(3)  Inoculate  four  such  tubes  with  -1  to  -5  cc.  of  the  sus- 
pected water,  and  place  them  in  the  warm  incubator  for 
twenty-four  hours. 

(4)  Then  test  one  tube  for  "  Cholera  Red,"  and  compare 
the  tint  with  that  obtained  from  a  tube  not  inoculated 
with  the  suspected  water :  the  former  should  be  a  deeper 
red ;   examine   another  tube   microscopically  for   vibrios ; 
from  the  others  make  gelatine  plates,  and  work  them  up  in 
the  manner  described  above. 

It  is  often  possible  to  give  a  definite  opinion  in  from 
eighteen  to  forty- eight  hours. 

III.  Agar-agar  plates. 

(1)  Liquefy  three  agar-agar  tubes  and  cool  them  down 
to  40°  C.  in  a  water  bath. 

(2)  Inoculate  one  with  -25  to  -5  cc.  of  the  suspected 
water. 

(3)  From  this   tube   inoculate  the  second   with   three 
platinum  loops,  and  from  this  second  tube  the  third  also 
with  three  loops. 


104  BACTERIOLOGICAL  ANALYSIS  LESSON  v 

(4)  Pour  plates  in  the  usual  manner,  and  when  the  agar- 
agar  is  firmly  set,  place  them  in  the  warm  incubator,  lid 
downwards,  so  that  the  condensation  water  does  not  collect 
on  the  surface  of  the  agar-agar. 

(5)  Examine  the  plates  next  morning  microscopically, 
and  make  subcultures  in  gelatine  (stab  cultures),  on  agar- 
agar  (streak  cultures),  and  in  peptone. 

Keep  the  gelatine  tubes  at  22°  C.,  the  agar-agar  and 
peptone  tubes  at  38 '5°  C. 

(6)  After  twenty -four  hours  examine  microscopically, 
and  test  the  peptone  tubes  for  "  Cholera  Ked." 

This  method  often  yields  quick  results. 
IV.  Gelatine  plates. 

(1)  Liquefy  three  gelatine  tubes  and  inoculate  the  first 
with  *25  to  *5  cc.  of  the  suspected  water. 

(2)  Proceed  exactly   as  in  the  case  of  the  agar-agar 
tubes  described  above. 

(3)  Prepare  three  plates  and  keep  them  at  22°  C. 

(4)  Examine  these  from  day  to  day  microscopically,  and 
also  make  subcultures  in  the  manner  stated  above. 

Unmistakable  colonies  will  be  found  after  thirty-six 
to  forty-eight  hours. 


LESSON  VI 

Examination  of  Milk— I.  Quantitative  Examination  by  Plate  Culture 
Method— II.  Qualitative  Examination— Bacillus  of  Typhoid  Fever 
— Bacterium  Coli  Commune— Streptococcus  Pyogenes— Bacillus 
Diphtherise— Tubercle  Bacillus 

Examination  of  Milk 

I.  QUANTITATIVE  EXAMINATION. 

As    ordinarily  obtained,  milk   always  contains   micro- 
organisms. 

(1)  Collect  the  milk  in  sterile  flasks,  having  first  shaken 
up  the  sample  supplied. 

(2)  With  a  sterile  pipette   inoculate   several  liquefied 
gelatine  tubes  with  quantities  varying  from  '005  cc.  to  "05 
or  '1  cc. 

(3)  Prepare  gelatine  plates,  and  place  them  in  the  cool 
incubator. 

(4)  Examine  and  count  the  colonies  in  the  same  manner 
as  was  described  on  p.  95. 

As  in  the  case  of  water,  so  here  also,  the  quantitative 
examination  is  of  comparatively  little  use,  the  quali- 
tative analysis  being  of  much  greater  importance. 


106  BACTERIOLOGICAL  ANALYSIS  LESSON 


II.  QUALITATIVE  EXAMINATION. 
Five  samples  of  milk  are  supplied. 

Sample  A  contains  Typhoid  bacilli. 

„       B         „       Bacterium  coli  commune. 
„       C        „       Streptococcus  pyogenes. 
„       D        „       Bacillus  diphtheriae. 
„       E        „       Tubercle  bacilli. 

(a)  Samples  A  'and  B :  Bacillus  of  typhoid  fever  and  the 
Bacterium  coli  commune. 

Examine  for  typhoid  bacilli  and  the  Bacterium  coli 
commune,  by  preparing  plates,  in  the  manner  described  on 
p.  105,  using,  however,  carbol  gelatine  instead  of  ordinary 
gelatine.  These  plates  must  be  worked  up  as  detailed  on 
p.  99.  The  nitration  method  is  unfortunately  not  applicable 
to  milk. 

(b)  Samples   C   and   D:   Streptococcus  pyogenes   and   the 
Bacillus  diphtherice. 

(1)  Use  three  sloped  agar-agar  tubes. 

(2)  Stir  up  the  milk,  so  as  to  distribute  the  suspended 
organisms  uniformly. 

(3)  Dip  a  stout  platinum  loop  into  the  milk. 

(4)  Now  make  three  parallel  streaks  on  the  surface  of 
the  first  agar-agar  tube. 

(5)  Without  dipping  the  needle  again  into  the  milk, 
make  three  parallel  streaks  on  the  surface  of  the  second 
agar-agar  tube,  and  also  of  the  third. 

(6)  Place    the    three    agar-agar    tubes    in    the    warm 
incubator. 


vi  TUBERCLE  BACILLI  IN  MILK  107 

Examine  next  day. 

Pick  out  the  smallest  colonies  which  appear  suspi- 
cious, and  make  subcultures  on  sloped  agar-agar, 
and  keep  the  latter  at  38'5°  C.  At  the  same 
time  make  a  microscopic  examination  of  the 
suspicious  colonies  (vide  p.  78). 

Streptococci  and  diphtheria  bacilli,  if  present  in 
sufficient  number,  can  readily  be  detected  in  this 
manner. 

If  diphtheria  bacilli  be  suspected,  instead  of  agar-agar, 
sloped  serum  agar-agar  may  be  used  with  great  advantage 
(vide  p.  92). 

(c)  Sample  E :  Tubercle  bacillus  (Van  Ketel's  method). 

(1)  To  200  cc.  of  the  suspected  milk  add  10  cc.  of  strong 
liquefied   carbolic   acid   or    10  grammes   of   carbolic    acid 
crystals. 

(2)  Shake  vigorously  in  a  well-corked  flask  for  two  to 
five  minutes. 

(3)  Now  pour  the  carbolised  milk  into  a  conical  urine 
glass,  and  allow  it  to  stand  under  a  glass  cover  for  twenty- 
four  hours. 

(4)  With  a  fine  capillary  pipette  remove  a  little  from 
the  deepest  layer  of  the  sediment,  and  prepare  films  in 
the  usual  manner,  by  rubbing  it  between  two  cover-glasses. 

(5)  Allow  the  films  to  dry  in  the  air  and  pass  them 
three  times  through  the  flame. 

(6)  Now  pass  the  films  through  a  solution  of  alcohol 
and  ether  (equal  parts). 


108  BACTERIOLOGICAL  ANALYSIS  LESSON  vi 

(7)  Dry  them  between  folds  of  blotting-paper  and  again 
pass  three  times  through  the  flame. 

(8)  Stain  in  carbol  fuchsine,  decolourise  in  hydrochloric 
acid    and    70   per    cent    alcohol,   and   counterstain   with 
methylene-blue,  as  described  on  p.  60. 

Mount  and  examine  with  -fa  in.  oil  immersion  for 
tubercle  bacilli. 


LESSON  VII 

Examination  of  Air  and  Dust — Plate  Culture  Method— Aspiration 
through  Broth — Filtration  through  Sugar 

Examination  of  Air  and  Dust 

I.  PLATE  CULTURE  METHOD. 

(1)  Prepare  three  gelatine  plates,  and  expose  them  to 
the  air  at  different  spots  and  levels  in  the  room  for  half  an 
hour,  one,  and  two  hours  respectively. 

(2)  Then  cover  them  up  with  sterile  lids,   and  place 
them  in  the  cool  incubator. 

(3)  Examine  the  plates  and  count  the  colonies   from 
day  to  day. 

The  plates  which  have  been  kept  nearest  the  floor, 
or  have  been  in  a  draught,  will  contain  most 
colonies.  The  number  of  colonies  will  also  vary 
with  the  time  of  exposure. 

Prepare  subcultures  in  gelatine  (streak  cultures)  of 
the  various  colonies. 

'  Various  kinds  of  Staphylococci,  Sarcinae,  and  Torulae 
or  Yeasts,  or  even  Cladothrix,  probably  will  be 
found. 


110 


BACTERIOLOGICAL  ANALYSIS 


LESSON 


This  is  a  rough  method  and  of  comparatively  little 
use. 

II.  ASPIRATION  THROUGH  BROTH. 

Put  up  an  apparatus  as  shown  in  the  diagram  (Fig.  6). 

(1)  Into  the  flask  (c)  pour  50 
cc.  of  sterile  beef  broth ;  close  it 
with  an  india-rubber  stopper  (5), 
and  pass  the  bent  tube  (a)  through 
the  perforated  stopper  into  the 
broth. 


A  spir> 


(2)  Now  plug  the  free  end  of 
the    bent    tube   and    of    e  with 


FIG.  6. 
a,  Bent  glass   tube   3  to  5  mm. 

in  diameter:  &,  india-rubber  T  -,  .-•. 

stopper ;c,  filtering  flask ;  d,   cotton-wool,    and    sterilise    the 
broth-  whole  apparatus  in  the  autoclave. 

(3)  When  the  flask  and  broth  have  cooled  down,  remove 
the  cotton-wool  plug  at  a  and  slowly  aspirate  air  through 
the  flask  by  means  of  the  air-pump. 

Continue  the  aspiration  for  half  an  hour  to  one  hour. 

(4)  Now  again  plug  a  with  sterile  cotton-wool. 

(5)  Liquefy  several  tubes  of  gelatine,  and  to  each  add 
•05  to  •!  cc.  of  the  broth  through  which  the  air  has  been 
aspirated,  and  prepare  plates  in  the  ordinary  manner. 

(6)  Keep  the  plates  in  a  cool  incubator ;  examine  them 
and  count  the  colonies  from  day  to  day. 

Colonies  should  be  examined,  both  microscopically 
and  by  means  of  subcultures,  as  they  appear. 

III.  FILTRATION  THROUGH  SUGAR. 

(1)  Powder  a  little  loaf  sugar  as  finely  as  possible. 


VII 


EXAMINATION  OF  AIR 


111 


(2)  Draw  out  a  glass  tube  (5  mm.  in  diameter  and  12  to 
15  cm.  in  length)  at  one  end  and  fuse  it  off  here. 

(3)  Fill  the  tube  with  the  powdered  sugar         A 
up  to  its  middle,  and  plug  its  open  end  with 

two  small   cotton -wool   plugs  (p1  and  j?2),  as 
shown  in  the  diagram  (Fig.  7). 

(4)  Sterilise  this  tube  in  the  hot-air  steriliser 
at  100°  C.  for  half  an  hour  to  one  hour. 

(5)  When  cool   remove  the   plug  p2,  and 
connect   the    sugar   tube  with  the  aspirating 
apparatus  (Fig.  8). 

(6)  Fix  the  sugar  tube  by  means  of  a  clamp 
(C1,  Fig.  9),  and  with  a  pair  of  sterile  forceps 
break  off  its  drawn-out  end. 

(7)  Loosen  the  clamp  of  the  aspirating  ap- 
paratus and  aspirate  by  alternately  changing  the  positions 

A 


Flo  7< 
and  *>2> sma11 

cotton -wool 

Plu§s- 


FIG.  8.— Aspirating  Apparatus. 


FIG.  9. 


of    bottles    I    and    II,    at    the   same    time   altering    the 
connections  as  required. 


112  BACTERIOLOGICAL  ANALYSIS          LESSON  vn 

Aspirate  for  one  or  two  hours,  counting  the  number 
of  aspirations  performed,  so  as  to  ascertain  the 
volume  of  air  taken  and  the  rapidity  of  aspiration. 

(8)  Now   remove   the   sugar  tube   and   shake  out  the 
sugar  into  a  flask  containing  50  cc.  of  broth,  which  of 
course  must  be  sterile. 

(9)  Liquefy  several  gelatine  tubes,  and  add  to  each  from 
•1  to  '5  cc.  of  the  sugar  solution,  and  pour  plates  in  the 
usual  manner;  and  work  them  up  as  before. 

Count  the  colonies  in  the  plates  in  the  way  described  on 
p.  95. 


LESSON  VIII 


Examination  of  Air  and  Dust  (conchided) — Anaerobic  Germs — Examin- 
ation of  Soil  —  Surface  Soil  —  Tetanus  and  Malignant  (Edema 
Bacilli — Anaerobic  Growth  in  an  Exhausted  Flask — Anaerobic 
Growth  in  Hydrogen — Fractional  Separation  of  Tetanus  Bacilli. 

Examination  of  Air  and  Dust  (concluded) 
IV.  ANAEROBIC  GERMS  IN  AIR  AND  DUST. 

(1)  Put   up  an   apparatus    as   shown   in   the   diagram 
(Fig.  10). 

(2)  Place  the  Wolff  bottle 
(A)  in  the  water  bath,  which 

mst    be    kept    steadily   at    Aspirator 
38-5°  C. 

(3)  Remove    the    cotton- 
wool plug   from   a,  and  by 

r  ,i  •  ,        ,       A,  Sterilised  Wolff  bottle  containing  50 

means  of  the  aspirator  slowly        to  100  cc.  of  sterile  liquid  grf  pe_ 

SUck    air    through   the    Warm  sugar  gelatine  ;  B,  water  bath  kept  at 

,      .         r        r    IP  i  38 '5°  C. ;  a  and  6,  tubes  plugged  with 

gelatine  tor  halt  an  hour  to        cotton-wool ;  c,  clamp  on  tube  con- 
one  hour  necting  apparatus  with  aspirator. 

(4)  Now  fuse   off  the   tube   at  /',  and  aspirate  again 
until  the  flask  (A),  which  must  be  kept  in  the  water  bath 
all  the  time,  is  exhausted. 

I 


FIG.  10. 


114  BACTERIOLOGICAL  ANALYSIS  LESSOX 

(5)  Clamp  at  C  as  tightly  as  possible,  so  as  to  prevent 
air  from  entering  into  the  flask  (A). 

It  is  advisable  to  smear  all  the  fittings  and  joints 
and  the  end  of  the  india-rubber  tube  below  C  with 
hard  paraffin. 

(6)  Place  the  flask  in  the  cool  incubator  and  watch  for 
the  appearance  of  colonies  in  the  gelatine.     If  such  appear, 
carefully  melt  the  gelatine  at  38°  C.  and  shake  the  flask 
gently,   and  with   a   sterile   pipette  fill  each   of   5  to  10 
tubes  with  10  cc.  of  the  gelatine. 

(7)  Prepare  roll  tubes  of  them  in  the  ordinary  manner. 
These  should  be  loosely  plugged. 

(8)  Place  these  tubes  in  a  sterilised,  large,  wide-necked 

bottle  containing  some  freshly  prepared 
solution  of  pyrogallic  acid  in  caustic 
potash1  (Fig.  11).  A  little  moistened 
sand  (s)  should  previously  be  placed  in 
the  flask,  for  the  tubes  to  rest  on. 

(9)    Close  the  wide  neck  of  the  flask 
with  a  perforated  india-rubber  stopper  (i) 
and  insert  a  bent  tube  (t)  through  the 
stopper,  which  must  not  extend  far  into  the  bottle. 

(10)  Connect   the   bent   tube  with   the   air-pump  and 
exhaust  the  bottle  thoroughly. 

(11)  When  all  the  air  has  been  exhausted,  clamp  tightly 

This  is  made  by  using  10  cc.  of  a  10  per  cent  solution  of  caustic 
potash  for  every  gramme  of  pyrogallic  acid  required.  The  caustic 
potash  solution  must  not  be  added  to  the  pyrogallic  acid  until  just 
before  closing  the  flask. 


vni  EXAMINATION  OF  SOIL  115 

at  C,  and  place  the  nask  with  the  roll  tubes  in  the  cool 
incubator  for  several  days. 

(12)  Then  examine  the  tubes  for  colonies,  and  make  sub- 
cultures in  grape-sugar  gelatine  (stab  cultures),  and  place 
them  in  a  wide-necked  bottle,  and  proceed  as  just  described. 

This  method  is  fairly  convenient  as  a  means  of  separ- 
ating anaerobic  organisms. 

Examination  of  Soil 
A.  SURFACE  SOIL. 

(1)  Scrape  a  little  earth  from  the  surface  of  the  ground 
with  a  sterile  knife  or  spoon,  and  collect  it  in  a  sterile 
beaker. 

(2)  Introduce  small  quantities  of  it  into  a  number  of 
liquid  gelatine  tubes  with  a  platinum   loop,  and   gently 
shake  the  tubes,  so  as  to  distribute  the  particles  of  earth  as 
uniformly  as  possible. 

(3)  Prepare  dilutions  in  the  usual  manner. 

(4)  Make  roll  tubes  as  previously  described,  and  place 
some  of  these  in  the  cool  incubator. 

Examine  the  tubes  from  day  to  day,  and  prepare  sub- 
cultures from  the  colonies  as  they  appear. 

(5)  Place  the  remaining  roll  tubes  in  a  large  wide-necked 
bottle  as  described  in  the  previous  section  (vide  Fig.  10) ; 
and  having  exhausted  the  flask,  place  it  in  the  cool  in- 
cubator. 

Examine  the  tubes  from  day  to  day,  and  prepare  sub- 
cultures in  grape-sugar  gelatine  (deep  stab  cultures). 


116  BACTERIOLOGICAL  ANALYSIS  LESSON 

The  latter  should  be  placed  in  a  wide-necked  bottle, 
and  this  should  be  exhausted  of  its  air. 

Every  time  the  bottle  is  opened  it  must  be  again 
exhausted. 

B.  EXAMINATION  OF  SOIL  FOR  TETANUS  AND  MALIG- 
NANT (EDEMA  BACILLI. 

Two  samples  of  sterilised  black  garden  earth,  artificially 
impregnated  with  tetanus  and  malignant  oedema  bacilli 
respectively,  are  supplied. 

(a)  Growth  in  an  exhausted  flask. 

(1)  Introduce  small  quantities  of  each  sample  into  tubes 
containing  liquid  grape-sugar  gelatine,  and  gently  shake 
the  tubes. 

(2)  Make  several  dilutions  in  the  ordinary  manner. 

(3)  Prepare  roll  tubes. 

(4)  Place  some  of  these  roll  tubes  in  a  wide -necked 
bottle,  which  must  then  be  exhausted  (vide  p.  114). 

(5)  Proceed  exactly  as  described  previously  (vide  p.  114). 

The  tubes  must  be  examined,  from  day  to  day,  for 
colonies  of  tetanus  or  malignant  oedema  bacilli. 

If  any  colonies  of  tetanus  or  malignant  oedema  bacilli 
appear,  start  subcultures  in  grape-sugar  gelatine 
(stab  cultures),  which  must  be  placed  in  a  flask, 
the  air  of  which  is  subsequently  exhausted. 

(b)  Growth  in  hydrogen. 

(I)  The  remaining  roll  tubes,  loosely  plugged,  should 


VIII 


ANAEROBIC  GROWTH  IN  HYDROGEN 


117 


be  placed  in  a  wide -necked  flask  or  bottle,  arranged  as 
shown  in  Fig.  12. 

(2)  Connect  the  flask  with  a  hydrogen  apparatus,  and 
allow  hydrogen  to  pass  through  it  for  one  hour. 

,  The  hydrogen  should  be  sent  through  three  wash 
bottles,  containing  respectively  lead  nitrate  (1:10), 
nitrate  of  silver  (1 : 10),  and  pyrogallic  acid  (added 
to  1  per  cent  caustic  potash)  solutions,  in  order  to 


FIG.  12. 
I.  II.  III.  Wash  bottles  ;  s,  sand  ;  p,  pyrogallic  acid  and  caustic  potash. 

remove  impurities,  such  as  arseniuretted  or  sul- 
phuretted hydrogen  (Fig.  12,  I.  II  III.) 

(3)  When  the  air  has  been  replaced  by  the  hydrogen — 
which  is  best  tested  by  applying  a  light  to  the  tube  a — 
fuse  off  at  a  first,  and  then  also  at  b,  and  place  the  flask 
in  the  cool  incubator. 

Examine  the  tubes  from  day  to  day,  and  work  them 
up  as  already  described,  i.e.  make  subcultures  in 
grape-sugar  gelatine  (stab  cultures). 

These  must  be  placed  in  a  wide-necked  bottle,  through 
which  hydrogen  is  again  passed  as  before. 


118 


BACTERIOLOGICAL  ANALYSIS 


LESSON 


Each  time   the  flask  is  opened   hydrogen  must  be 
passed  through  it  again. 

(c)  Fractional  separation  of  tetanus  bacilli. 

(1)  Introduce  small  quantities  of  the  earth  impregnated 
with  tetanus  bacilli  into  three  tubes  containing  liquid  grape- 
sugar  agar-agar,  and  also  into  three  tubes  containing  liquid 
grape-sugar  gelatine,  kept  at  60°  C. 

(2)  Heat  them  for  half  an  hour  at  60°  C.,  and  then  allow 
them  to  cool. 

(3)  Now  place  them  in  a  flask,  which  must  be  exhausted 
in  the  manner  already  described  (vide  p.  114). 

(4)  Place  flask  and  tubes  in  the  warm  incubator  for 
twenty-four  hours. 

(5)  Now  heat  the  tubes  again  at  60°  C.  for  half  an  hour 
to  one  hour ;  and  place  them  in  a  flask,  exhausted  of  air,  in 

the  warm  incubator. 

(6)  Examine  the  tubes  on  the 
second  or  third  day  by  means  of 
films ;  and  if  the  cultures  appear  to 
be  pure,  make  subcultures  in  grape- 
sugar  agar-agar,  and  in  grape-sugar 
gelatine  (deep  stab  cultures). 

These  must  be  placed  in  a 
flask,  which  then  must  be 
exhausted  and  placed  in  the 
warm  or  cool  incubator,  as 
the  case  may  be. 

If  the  cultures,  however,  are  not,  or  appear  not  to  be, 
pure,  the  tubes  should  be  heated  once  more  at  60°  C.  for 


FIG.  13. 

a,  Thermometer ;  6,  tliermo- 
regulator. 


viii  SEPARATION  OF  TETANUS  BACILLI  119 

half  an  hour  to  one  hour,  as  above  described.     This  should 
be  repeated,  until  the  cultures  are  pure. 

The  separation  of  tetanus  bacilli  without  animal 
inoculations  is  by  no  means  easy. 

The  gelatine  cultures  when  pure  may  be  kept  in  the 
cool  incubator. 

To  keep  the  tubes  at  a  steady  temperature  of  60°  C. 
arrange  a  large  beaker,  filled  with  water,  over  a 
gas  flame,  and  regulate  the  temperature  by  means 
of  a  thermo-regulator,  as  shown  in  the  diagram 
(Fig.  13). 


LESSON  IX 

Examination  of  Decomposing  Meat — Aerobic  Putrefaction — Anaerobic 
Putrefaction — How  to  Examine  a  Sample  of  Unsound  Meat — How 
to  Examine  Meat  for  Trichina  Spiralis — Cysticercus — Psorosper- 
mosis — Examination  of  Ice  Cream. 

Examination  of  Decomposing  or  Diseased  Meat 

A.  PUTREFACTION. 

Mince  a  little  fresh  meat,  and  add  small  quantities  of 
it  to  five  tubes  containing  sterile  nutrient  broth. 

(a)  Aerobic  putrefaction. 

(1)  Place  two  tubes  in  the  warm  incubator. 

(2)  After  three  to  four  days  examine  the  contents  of  the 
tubes  microscopically  by  means  of  cover-glass  films,  and 
prepare  plates  from  each  in  the  usual  manner. 

(3)  Work  up  the  colonies  as  they  appear. 

Various  kinds  of  Proteus,  amongst  others  the  Proteus 
Zenkeri,  Bacillus  coli  communis,  Torulse,  Staphylo- 
cocci,  Sarcinse,  etc.,  will  be  found. 

(b)  Anaerobic  putrefaction. 

(1)  Place  two  of  the  tubes  in  a  flask,  which  should  be 
exhausted  of  air  in  the  manner  already  described. 


LESSON  ix  PUTREFACTIVE  ORGANISMS  121 

(2)  After  three  to  four  days  examine  these  tubes  and 
compare  them  with  the  aerobic  tubes. 

The  odour  will  generally  be  more  marked. 

(3)  Examine  the  contents  of  the  tubes  microscopically, 
and  then  prepare  roll  tubes  from  them  in  the  ordinary 
manner,  and  place  them  in  a  flask  exhausted  of  air,  which 
must  be  kept  in  the  cool  incubator. 

(4)  Work  up  the  colonies  as  they  appear,  and  make  sub- 
cultures in  grape-sugar  gelatine  (deep  stab  cultures). 

(5)  Place  these  in  an  exhausted  flask  kept  at  22°  C. 

Streptococci,  Bacillus  coli  communis,  and  other  bacilli 
will  be  found. 

(c)  Control  experiment. 

(1)  From  the  last  or  fifth  tube,  while  fresh,  prepare 
three  gelatine  plates,  and  also  three  gelatine  roll  tubes,  in 
the  usual  manner. 

(2)  Put  the  plates  in  the  cool  incubator,  and  work  up  the 
colonies  in  the  ordinary  way. 

(3)  Keep  the  roll  tubes  in  the  cool  incubator  in  an 
exhausted  flask,  and  work  up  the  colonies  in  exactly  the 
same  manner  as  described  above. 

Compare  these  results  with  the  previous  ones. 

(d)  Sterilisation  and  putrefaction. 

(1)  Take  a  little  fresh,   minced  meat,  and  add  small 
quantities  of  it  to  broth  tubes. 

(2)  Heat  these  in  the  autoclave,  in  order  to  sterilise  their 
contents. 


122  BACTERIOLOGICAL  ANALYSIS  LESSON 

(3)  Now  keep  them  at  38 '5°  C.  for  several  days. 
There  will  be  no  smell. 

Examine  the  contents  of  the  tubes  microscopically, 
and  make  plates. 

The  latter  should  remain  sterile. 

B.  HOW  TO  EXAMINE  A  SAMPLE  OF  UNSOUND  MEAT.1 

(1)  Feed  mice  or  rats  with  portions  of  it,  and  watch  the 
effect. 

If  they  die,  make  plate  cultures  from  the  organs  and 
heart's  blood  (vide  p.  42). 

(2)  Make  an  extract  in  sterile  broth  or  saline  solution  of 
the  sample  supplied,  and  inoculate  mice  or  other  animals 
subcutaneously. 

If  they  die,  make  plate  cultures  as  before,  and  separate 
the  organisms  obtained. 

(3)  Prepare  an  extract  as  above,  and  make  plate  cultures 
in  the  ordinary  way,  and  also  roll  tubes. 

Keep  the  former  aerobically,  the  latter  anaerobically. 
Separate  the  various  organisms  as  they  appear. 

In  all  cases,  the  organisms  which  are  separated  should 
be  examined  as  to  their  virulence,  both  by  means 
of  feeding  experiments  and  by  inoculations. 

In  cases  of  food  poisoning,  it  has  been  shown  that  at 
times  organisms  may  kill  if  taken  in  by  the  mouth, 
while  they  fail  to  do  so  if  injected  subcutaneously. 

For  separation  of  ptomaines  and  toxines  vide  p.  172. 

1   These  experiments  can  only  be  performed  under  a  licence  and  a 
special  certificate. 


ix  PARASITES  IN  MEAT  123 

C.  HOW  TO   EXAMINE   MEAT  FOR   TRICHINA   SPIRALIS. 

(1)  Place  a  small,  thin  piece  of  the  suspected  muscle  on 
a  strong  glass  slide,  and  press  another  slide  firmly  down  on 
to  the  meat,  in  order  to  flatten  it  out  in  a  uniform  layer. 

Examine  under  the  low  power  or  with  a  dissecting 
microscope  :  the  capsules  will  be  recognised  at  once 
as  small  white  points  lying  between  the  fibres. 

(2)  Tease  out  a  few  fibres  under  the  low  power  of  a 
dissecting  microscope,  and  carefully  separate  the  small  white 
bodies. 

Mount  in  Farrant's  solution,  and  examine  with  low 
and  high  powers. 

If  the  capsule  of  the  trichina  is  calcified,  add  a  few 
drops  of  10  per  cent  hydrochloric  acid  in  order  to 
dissolve  out  the  lime,  wash  and  drain  off  the  water, 
and  then  mount  in  Farrant's  solution. 

D.  EXAMINATION  or  CYSTICERCUS  OF  EABBIT. 

(1)  Carefully  open  the  small  cyst,  and  tease  out  its  wall 
in  a  drop  of  water  under  a  dissecting  microscope. 

(2)  Search  for  the  head  or  scolex,  and  free  it  as  much  as 
possible  from  its  attachments. 

(3)  Drain  off  the  superfluous  water  with  a  piece  of  blot- 
ting-paper. 

(4)  Mount  in  Farrant's  solution,  applying  gentle  pressure 
bo  the  cover-glass,  so  as  to  flatten  out  the  head. 

Examine  under  a  low  power. 
Notice  the  suckers  and  hooks. 


124  BACTERIOLOGICAL  ANALYSIS  LESSON 

E.   PSOROSPERMS   IN  RABBIT'S  LlVER. 

(a)  Examination  in  the  v/nstained  condition. 

(1)  Make  a  few  cuts  into  the  liver  and  look  for  white, 
puriform  or  caseous  masses. 

(2)  Remove   some    of    the   white   substance   on   to   a 
slide,  and  tease  it  out  carefully. 

(3)  Add  a  drop  or  two  of  a  10  per  cent  caustic  potash 
or  a  little  iodine  solution,  and  allow  either  to  act  for  a  few 
minutes. 

(4)  Remove  the  excess  of  caustic  potash  or  iodine,  and 
then  mount  the  specimen  in  Farrant's  solution. 

Examine  with  low  and  high  powers,  using  a  narrow 
diaphragm  at  the  same  time. 

(b)  Staining  of  psorosperms. 

(1)  Squeeze  some   of  the  white  matter  between  two 
cover-glasses,   and  when  dry  pass  the  films  through  the 
flame. 

(2)  Stain  them  in  Lofner's  methylene-blue,   or  as   for 
tubercle  bacilli,  in  carbol-fuchsine  and  methylene-blue. 

In  the  latter  case  the  psorosperms  will  appear  red. 

Examination  of  Ice  Cream 

(a)  Quantitative  examination. 

(1)  Melt  some  ice  cream  (100  cc.)  in  a  sterile  beaker  at 
38°  C.  and  add  500  cc.  of  sterile  distilled  water  to  it. 

(2)  Prepare  plates  from  the  mixture,   using   ordinary 
gelatine  and  also  carbolic  acid  gelatine,  inoculating  the  tubes 
with  quantities  varying  from  '005  to  '05  cc.  (vide  p.  93). 


ix  BACTERIA  IN  ICE  CREAM  125 

(3)  Keep  the  plates  at  the  ordinary  temperature,  count 
the  colonies,  and  work  them  up  in  the  ordinary  manner, 
both  microscopically  and  by  means  of  subcultures  in 
gelatine. 

(b)  Examination  for  typhoid  bacilli  and  the  Bacterium  coli 
commune. 

(1)  If   it  is   desirable   to  test    ice    cream  for  typhoid 
bacilli  or  the  Bacterium  coli  commune,  a  large  quantity  of 
sterile  water  must  be  added,  for  otherwise  the  plates  will 
be  over-grown. 

(2)  Work  these  up  in  the  same  manner  as  described  on 
p.  106. 

The  Bacterium  coli  commune  is  frequently  found  in 
ordinary  street  ices. 


LESSON   X 

Examination  of  Antiseptics  and  Disinfectants — Method  of  Testing 
Antiseptics — Methods  of  Testing  Disinfectants — Koch's  Method — 
Carbolic  Acid — Mercuric  Chloride — Steinberg's  Method  of  Testing 
Antiseptics — Disinfectant  Action  of  Gases — Sulphur  Dioxide — 
Chlorine — Ammonia. 

Examination  of  Antiseptics  and  Disinfectants 

A.  METHOD  OF  TESTING  ANTISEPTICS. 

Carbolic  acid. 

(1)  Prepare  a  series  of  carbolic  acid  broth  tubes  (1 : 100, 
1  :  200,  1  :  300,  1  :  400,  1 :  500). 

To  tubes  containing  10  cc.  of  broth  add  respectively, 
with  a  sterile  graduated  pipette,  *1  cc.,  '05  cc., 
•03  cc.,  "025  cc.,  '02  cc.  of  liquid  carbolic  acid. 

(2)  Inoculate  these  five  tubes  with  a  platinum-loopful  of 
a  pure  culture  in  broth  of  the  bacillus  of  typhoid  fever  or 
blue  pus,  twenty-four  hours  old. 

Inoculate  also  an  ordinary  broth  tube  with  the  same 
organisms,  for  the  purpose  of  control. 

(3)  Place  the  six  tubes  in  the  warm  incubator. 
Examine  and  compare  the  tubes  from  day  to  day. 


LESSON  x  TESTING  OF  DISINFECTANTS  127 

B.  METHODS  or  TESTING  DISINFECTANTS. 

(i.)  Koch's  method  (modified). 

(a)  Test  the  following  solutions  : 

Absolute  alcohol ; 
Mercuric  chloride,  1  : 1000  ; 
Mercuric  iodide,  1  :  2000  ; 
Carbolic  acid,  1  : 20  ; 
Condy's  fluid. 

(1)  Keep  anthrax  silk  threads1  in  these  solutions  for 
equal  periods  of  time,  varying  from  two  to  twenty-four 
hours. 

(2)  Then  take  them  out  of  the  fluid  with  sterile  forceps 
or  needles. 

(3)  Wash  the  threads  in  sterile  water ;  and  do  the  same 
with  a  thread  that  has  not  been  kept  in  a  disinfecting 
solution. 

(4)  Now  press  the  threads  into  the  substance  of  an  agar- 
agar  plate,  which  must  be  kept  at  38 '5°  C.     Small  labels 
must  be  fastened  on  the  lid  of  the  Petri's  capsule,  in  order 
to  avoid  confusion. 

Examine  the  plate  from  day  to  day. 

1  Anthrax  silk  threads  are  prepared  by  making,  under  strictly 
aseptic  precautions,  a  suspension  in  broth  or  "6  per  cent  saline  solution 
of  virulent  agar-agar  or  potato  cultures  of  anthrax  bacilli,  grown  at 
38'5°  C.  for  several  days,  and  known  to  consist  almost  entirely  of 
spores. 

Sterilised  silk  threads,  about  an  inch  in  length,  are  allowed  to  soak 
in  this  suspension  for  half  an  hour  to  one  hour,  are  then  collected  in  a 
sterile  capsule,  and  dried  in  the  warm  incubator. 


128  BACTERIOLOGICAL  ANALYSIS  LESSON 

It  will  be  found  that  mercuric  iodide  is  more  germi- 
cidal  than  mercuric  chloride,  and  that  absolute 
alcohol  and  Condy's  fluid  have  but  little  or  no 
effect  on  the  anthrax  spores. 

(&)  Test  the  following  solutions  of  carbolic  acid  : 

in  alcohol,  1  :  20  ; 
in  glycerine,  1:20; 
in  water,  1  :  20  ; 

in  water,  1:25,  with  2  to  4  per  cent  of  hydro- 
chloric acid. 

Proceed  exactly  as  above,  allowing  the  threads  to 
soak  in  the  solutions  for  at  least  four  hours. 

It  will  be  found  that  a  solution  of  carbolic  acid  in 
weak  hydrochloric  acid  is  very  efficacious,  while 
solutions  in  glycerine  and  alcohol  are  of  little  use. 


(c)  Mercuric  chloride 

(1)  Keep  anthrax  threads  in  this  solution  for  two  to 
four  hours. 

(2)  Now  wash  some  of  them  in  sterile  water  ;  wash  the 
others  first  in  a  solution  of  ammonium  sulphide  and  then 
in  water. 

(3)  Fix  the  threads  on  an  agar-agar  plate,  and  place  the 
latter  in  an  incubator  at  38  '5°  C. 

Examine  the  threads  from  day  to  day. 

Growth  will  be  observed  around  the  threads  washed 
with  ammonium  sulphide,  while  around  the  others 
there  is  either  no  growth  at  all  or  very  limited 
growth 


x  DISINFECTION  WITH  GASES  129 

(ii.)  Sternberg's  method. 

This  method  is  more  convenient  than  Koch's  method  for 
all  practical  purposes,  and  the  results  are  more  useful, 
because  the  conditions  of  the  experiment  resemble  more 
closely  those  of  practice. 

(1)  Take  five  tubes  containing  5  cc.  of  broth  each,  and 
inoculate  them  with  the  bacillus  of  typhoid  fever. 

(2)  After  twenty-four  hours  add  to  four  of  the  tubes 
respectively  5  cc.  of  carbolic  acid  solutions,  of  the  following 
strengths,   1  :  200,   1  :  100,   1  :  50,   1  :  20.      The  fifth  tube 
must  be  left  for  the  purpose  of  control. 

(3)  Place  the  tubes  in  the  warm  incubator  for  two  to  four 
hours,  or  other  periods  of  time. 

(4)  Then  make  five  subcultures   in   broth   from   these 
five  tubes  (one  or  two  platinum-loopfuls). 

(5)  Keep  them  in  the  incubator  at  38 -5°  C.  for  several 
days. 

Examine  the  tubes  for  growth  from  day  to  day. 
(The  carbolic  acid  solutions  must  be  prepared  with 
sterilised  water.) 

(C)  DISINFECTANT  ACTION  OF  GASES. 
(a)  Sulphur  dioxide. 

(1)  Dip  strips  of  sterilised  cloth  in  a  fresh  broth  cul- 
ture of  Staphylococcus  pyogenes  aureus,  place  them  in  a 
sterile  Petri's  capsule,  and  allow  them  to  dry  in  the  warm 
incubator. 

(2)  Fix  up  an  apparatus  in  the  fume  cupboard  to  pre- 
pare sulphur  dioxide,  as  shown  in  the  diagram  (Fig.  1 4). 

K 


130 


BACTERIOLOGICAL  ANALYSIS 


LESSON 


(3)  Allow  the  gas  to  pass  over  one  of  the  strips  of  cloth 
for  one  to  two  hours. 


Fia.  14.— Flask  I  contains  some  copper  and  sulphuric  acid,  which  are  heated  over 
the  flame  to  prepare  the  sulphur  dioxide  ;  II,  Wash  bottle  containing  water  ; 
III,  Wide  glass  tube  in  which  the  infected  strip  of  cloth  is  placed  ;  IV,  Spiral 
glass  tube  surrounded  by  a  freezing  mixture  of  salt  and  pounded  ice ;  V, 
Small  flask,  placed  in  a  beaker  containing  ice  and  salt. 

(4)  Then   remove   it   and   drop  it  into   a  broth  tube, 
which  is  to  be  kept  at  38 '5°  C.  for  several  days. 


FIG.  15.— I,  Flask  containing  sodium  chloride  (one  part  by  weight),  manganese 
dioxide  (one  part  by  weight),  and  sulphuric  acid  and  water  (two  parts  by 
weight  of  each),  which  must  be  very  gently  heated  ;  II,  Wash  bottle  containing 
water ;  III,  Wide  glass  tube  containing  the  infected  strip  of  cloth ;  IV, 
Collecting  cylinder  filled  with  water. 

Examine  the  contents  of  the  tube  for  growth  from  day 
to  day,  both  microscopically  and  by  means  of  plates. 


x      DISINFECTION  WITH  CHLORINE  AND  AMMONIA     131 


(6)  Chlorine. 

(1)  An  apparatus  similar  to  the  one  just  described  may 
be  fixed  up,  as  shown  in 

diagram  (Fig.  15). 

(2)  Allow  the  chlorine 
to  pass  over  one  of   the 
strips  of  cloth  for  half  an 
hour  to  one  hour. 

(3)  Then    remove    it, 
and  place   it   in   a  broth 
tube  to  be  kept  at  3 8 '5°  C. 

Examine  it  for 
growth  from  day 
to  day  as  before. 


(c)  Ammonia. 


FIG.  16. — I,  Flask  containing,  a,  mixture  of 
sal-ammoniac  (one  part  by  weight)  and 
quicklime  (two  parts  by  weight);  5,  a 
layer  of  powdered  quicklime ;  the  flask 
with  its  contents  is  heated  over  a  Bunsen 
flame ;  II,  Wide  glass  tube  containing 
the  infected  strip ;  III,  Flask  containing 
water  to  receive  the  ammonia. 

(1)  Fix  up  an  apparatus  as  shown  in  the  accompanying 
diagram  (Fig.  16). 

(2)  Allow  the  ammonia  to  pass  over  one  of  the  strips  of 
cloth  for  half  an  hour  to  one  hour,  and  proceed  as  before. 

Examine  the  broth  tube  for  growth  from  day  to  day. 


LESSON  XI 

Examination  of  an  Animal  dead  of  a  Bacterial  Disease — Anthrax — 
Pyocyaneus  Septicsemia — Cholera  Asiatica — Rapid  Method  of 
Embedding  Tissues  in  Paraffin — Examination  of  Typhoid  Spleen. 

How  to  Examine  an  Animal  dead  of  Bacterial  Disease 
Three  animals  are  supplied  : 

(a)  mouse  inoculated  with  Bacillus  anthracis ; 

(&)  guinea-pig     inoculated     intraperitoneally    with 
Bacillus  pyocyaneus ; 

(c)  guinea  -  pig   inoculated     intraperitoneally    with 
Vibrio  choleras  Asiatics. 

(1)  Nail  the  animal  out  on  a  wooden  board,  thoroughly 
washed  with  mercuric  chloride  1 : 1000. 

(2)  Moisten  the  hairy  surface  of  the  animal's  abdomen 
with  spirit,  and  then  wash  it  with  mercuric  chloride  1  : 1000. 

(3)  With  a  pair  of  sterilised  forceps  and  a  pair  of  sterilised 
scissors  carefully  reflect  the  skin  from  the  abdomen  and 
chest. 

(4)  Thoroughly  cauterise  the  exposed  surface  with  a  red- 
hot  glass  rod,  and  then  open  the  chest  and  abdominal  cavity 
with  sterile  instruments 


LESSON  xi       CULTIVATIONS  FROM  DEAD  ANIMALS  133 

(a)  Prepare  three  gelatine  plates  severally  from  the 
spleen,  peritoneal  fluid,  and  heart's  blood  in  the  ordinary 
manner,  and  place  them  in  the  cool  incubator. 

(6)  Also  make  agar-agar  streak  cultures  from  the  spleen 
of  the  mouse,  and  from  the  peritoneal  fluid  of  the  guinea- 
pigs  (vide  p.  106). 

Place  these  in  the  warm  incubator. 

The  plates  and  agar-agar  tubes  should  be  worked  up 
in  the  usual  manner. 

(c)  Prepare  also  cover-glass  specimens  of  the  spleen  and 
peritoneal  fluid,  and  stain  them  with  Loffler's  methylene- 
blue,  and  examine  them  with  -  in.  oil  immersion. 


Quick  Method  of  Hardening  and  of  Embedding 
in  Paraffin 

(1)  Place  small  pieces  of  the  mouse's  spleen,  kidney, 
liver,  and  lung  in  absolute  alcohol. 

(2)  Change  the  alcohol  every  half -hour,  using  always  an 

excess. 

(3)  After  one  and  a  half  to  two  hours  place  the  small 
pieces  of  tissue  in  a  small  corked  bottle  containing  benzol, 
till  they  are  transparent  (a  few  minutes). 

(4)  Now  pour  off  the  excess  of  benzol,  so  that  the  pieces 
are  only  just  covered  by  it,  add  a  few  shavings  of  paraffin 
(56°  to  60°  C.  melting  point),  and  place  the  bottle  on  the 
paraffin  stove,  till  the  paraffin  liquefies. 


134  BACTERIOLOGICAL  ANALYSIS  LESSON 

(5)  Transfer  the  specimens  to  liquid  hard  paraffin  (56° 
to  60°  C.),  and  allow  them  to  soak  for  five  minutes. 

(6)  Pour  the  paraffin  and  the  tissues  into  a  small  paper 
box,  and  when  the  paraffin  is  set,  cut  out  the  pieces  of 
tissue,  fix  them   on  the   rocking  microtome,  and   cut  a 
number  of  sections,  which  must  be  received  in  warm  water 
at  60°  to  65°  C. 

(7)  Fix  the  sections  on  cover-glasses  and  dissolve  off  the 
paraffin  in  the  usual  manner  (vide  p.  64). 

(8)  Now  stain  the  specimens  fixed  on  the  cover-glasses : 

(a)  according  to  Gram's  method,  as  described  on  pp. 
70  and  71 ; 

(b)  with  methylene-blue  for  five  minutes ;   wash  in 
water  for  half  a  minute,  then  in  water  acidulated 
with  acetic  acid  for  half  a  minute,  and  again  in 
water  for  half  a  minute ;   dehydrate  in  alcohol, 
clear  and  mount  (vide  p.  35). 

Examine  the  sections  under  the  microscope. 

In  this  manner  good  specimens  can  be  obtained  in  less 
than  three  hours. 


Slow  Method  of  Hardening  Tissues 

(1)  Pieces  of  tissues  from   the  mouse  also  should  be 
placed  for  a  few  weeks  in  Miiller's  fluid,  which  must  be 
changed  from  time  to  time. 

(2)  Then  wash  the   specimens   in  running  water,   to 
remove  the  Miiller's  fluid. 


xi  HAKDENING  OF  TISSUES  135 

(3)  Keep  the  washed  tissues  in  methylated  spirit  for  a 
week,  and  then  place  them  in  absolute  alcohol. 

(4)  Now  embed  in  paraffin  (56°  to  60°  0.)  in  the  ordinary 
and  slower  manner,  cut  and  stain. 

Examine  the  sections,  and  compare  them  with  the 
others. 


Examination  of  Typhoid  Spleen 

(1)  Wash  the  surface  of  the  spleen  with  sterilised  water 
and  cauterise  a  small  area  with  a  red-hot  glass  rod. 

(2)  Thrust  a  sterilised  stout  platinum  loop  through  the 
cauterised  spot  well  into  the  spleen,  and  gently  stir  up  the 
pulp. 

(3)  Withdraw  the  needle  and  inoculate   three   sloped 
gelatine  tubes,  as  described  for  the  Bacillus  diphtherise  on 
p.  106. 

(4)  Place  the  tubes  in  a  cool  incubator  and  examine 
them  next  morning. 

In  most  cases  pure  cultures  are  readily  obtained  from 
a  typhoid  spleen. 

To  confirm  the  diagnosis,  sub-cultures  must  be  made 
in  milk,  broth,  gelatine  (stab  and  shake) ;  broth 
cultures  (six  to  twelve  days'  old)  must  be  tested 
for  Indol  and  young  agar-agar  cultures  stained  for 
flagella  (vide  pp.  38  and  46). 


LESSON  XII 

Testing  of  Filters — Filtering  through    Paper — Filtering  through  a 
Berkefeld  Filter— The  Effect  of  Use  on  a  Berkefeld  Filter. 

Testing  of  Filters 

A.  FILTERING  THROUGH  PAPER. 

(1)  Take  500  to  1000  cc.  of  sterile  water,  and  add  three 
broth  cultures  of  the  Bacillus  prodigiosus  (four  days  old). 

(2)  Prepare  three  gelatine  plates  from  this  water,  before 
filtering  it. 

(3)  Now  filter  the  water  through  sterile  paper,  using  a 
sterile  funnel  and  observing  aseptic  precautions  as  strictly 
as  possible. 

(4)  Prepare  three  gelatine  plates  from  the  filtrate. 
Place  the  two  sets  of  plates  in  the  cool  incubator  and 

compare   them   from   day   to   day,  counting  the 
colonies  at  the  same  time. 

Filter  paper  arrests  only  a  limited  number  of  organ- 
isms. 

B.  FILTERING  THROUGH  A  BERKEFELD  FILTER. 

(1)  Take  1000  cc.  of  sterile  water,  and  add  three  broth 
cultures  of  the  Bacillus  prodigiosus  as  before. 


LESSON  xii  EFFECT  OF  FILTRATION  137 

(2)  Filter  through  a  sterile  Berkefeld  filter  into  a  sterile 
flask. 

(3)  Prepare  three  gelatine  plates  from  the  filtrate,  and 
place  them  in  the  cool  incubator. 

Examine  them  from  day  to  day. 
•The  plates  should  remain  sterile. 

C.  EFFECT  OF  USE  ON  A  BERKEFELD  FILTER. 

(1)  Take  1000  cc.  of  sterile  water,  and  add  three  broth 
cultures  of  the  Bacillus  prodigiosus  as  above. 

(2)  Filter  through  a  sterile  Berkefeld  filter  into  a  sterile 
flask. 

(3)  Pour  the  filtrate  back  into  the  filtering  cylinder  and 
filter  again. 

(4)  Repeat  this  process  six  times. 

(5)  Eventually  pour  the  filtrate  back  into  the  cylinder 
and  allow  the  filter  to  stand  for  a  day. 

(6)  Eepeat  the  whole  process  with  the  same  filter  on 
three  successive  days,  without  cleaning  it  out  in  the  mean- 
time 

(7)  On  the  last  day  fix  a  fresh  sterile  flask  to  the  filter, 
and  exhaust  it  with  the  air-pump. 

(8)  From  the  final  filtrate  three  gelatine  plates  should 
be  prepared  in  the  usual  manner. 

Examine  them  from  day  to  day. 

The  plates  will  show  colonies  of  the  Bacillus  pro- 
digiosus. 


138  BACTERIOLOGICAL  ANALYSIS         LESSON  xn 

(9)  Now  clean  the  "candle"  of  the  filter  thoroughly, 
scrubbing  it  with  a  brush,  and  sterilise  the  whole  apparatus 
in  the  autoclave. 

(10)  When  it  has  cooled  down,  filter  through  it  1000  cc. 
of  sterile  water,  to  which  three  broth  cultures  of  the  Bacillus 
prodigiosus  have  been  added. 

(11)  From  the   filtrate  prepare  three  gelatine   plates, 
which  should  be  kept  in  the  cool  incubator. 

Examine  them  from  day  to  day. 
The  plates  should  remain  sterile. 

The  experiment  proves  that  filters  at  best  can  do  only 
a  limited  amount  of  work,  and  must  be  cleansed 
or  sterilised  from  time  to  time. 

Other  filters  may  be  tested  in  the  same  manner, 


PART    III 
BACTERIOLOGICAL   CHEMISTRY 

LESSONS   I-X 


LESSON  I 

Preparation  of  Metabolic  Products  of  Micro-organisms — Heat — Filtra- 
tion— Combined  Heat  and  Filtration — "  Intracellular  "  Poisons — 
Nitrous  Acid  in  Cholera  Cultures. 

Preparation  of  Metabolic  Products  of  Micro-organisms 

(a)  Sterilisation  by  heat. 

(1)  Inoculate   twelve    broth    tubes   with  the   Bacillus 
pyocyaneus,  and  keep  them  at  38*5°  C.  for  a  week. 

(2)  Place  them  in  the  water  bath  at  70°  C.  for  ten  to 
fifteen  minutes. 

(b)  Sterilisation  by  filtration. 

(1)  Inoculate  several  small  flasks  of  broth  (containing 
50  cc.  each)  with  the  Bacillus  pyocyaneus,  and  keep  them 
at  38-5°  C.  for  a  week. 

(2)  Filter  the  broth  cultures  through  a  sterile  Berkefeld 
filter  into  a  sterile  flask. 

(c)  Combined  sterilisation  by  heat  and  filtration. 

(1)  Inoculate  several  small  flasks  of  broth  (containing 
50  cc.  each)  with  the  Bacillus  pyocyaneus,  and  keep  them 
at  38-5°  C.  for  a  week. 


142  BACTERIOLOGICAL  CHEMISTRY  LESSON 

(2)  Place  them  in  the  water  bath  at  70°  C.  for  fifteen 
minutes. 

(3)  Filter  the  sterilised  broth  cultures  through  a  sterile 
Berkefeld  filter  into  a  sterile  flask. 

(d)  "  Intracellular  "  poisons. 

(1)  Prepare  ten  agar-agar  streak  cultures  of  the  Bacillus 
prodigiosus,  and  keep  them  at  22°  C.  for  twenty-four  to 
forty-eight  hours.      The  whole   surface   of  the  agar-agar 
should  be  uniformly  inoculated. 

(2)  To  each  tube  add  10  cc.  of  sterile  '6  per  cent  saline 
solution,  and  with  a  stout  platinum  needle  carefully  scrape 
the  culture  off  the  agar-agar  into  the  salt  solution,  so  as  to 
prepare  suspensions  of  the  bacilli. 

(3)  Pour  the  liquid  contents  of  the  tubes  into  a  small 
flask. 

(4)  Heat  the  flask  and  its  contents  in  a  water  bath  at 
65°  to  70°  C.  for  fifteen  minutes. 

(e)  Filtered  intracellular  poisons. 

Proceed  as  above,  but  conclude  by  filtering  the  sterilised 
contents  of  the  flask  through  a  sterile  Berkefeld  filter  into 
a  sterile  flask. 

In  every  case  the  solution,  after  sterilisation  by  heat  or  jiltra- 
tion,  must  he  tested  by  inoculating  from  it  two  agar-agar  tubes 
(streak  cultures).1 

1  If  the  agar-agar  tubes  show  any  growth,  the  solutions  must  be 
sterilised  again,  by  heat  or  filtration,  as  the  case  may  be. 


i  TESTING  FOR  NITROUS  ACID  143 

Test  for  Nitrous  Acid  in  Cultures 

(1)  Filter   six  broth   cultures   of   the   Vibrio   choleras 
Asiaticse  (two  days  old)  through  a  small,  sterile  Berkefeld 
filter  into  a  sterile  flask. 

(2)  To  a  portion  of  the  filtrate  add  a  drop  or  two  of 
hydrochloric  acid,  and  then  a  solution  of  the  hydrochloric 
acid  salt  of  meta-phenylenediamine. 

The  solution  will  become  yellowish  red  or  deep  red, 
according  to  the  amount  of  nitrous  acid  (nitrites) 
present  (Griess's  reaction). 

The  red  colour  is  due  to  the  formation  of  phenylene 
brown  or  Bismarck  brown. 

Test  broth  cultures  of  the  Proteus  vulgaris  in  the  same 
manner. 


LESSON  II 

Protei'nes — Precipitation  of  Proteines  by  Alcohol — Bacterial  Extracts. 

Proteines  (Nencki,  Buchner,  R6mer) 

(a)  Bacillus  pyocyaneus. 

(1)  Inoculate  twenty  agar-agar  tubes  with  the  Bacillus 
pyocyaneus,  and  keep  them  at  38*5°  C.,  until  there  is  a 
copious  growth  over  the  whole  surface  of  the  agar-agar. 

(2)  Into  each  tube  pour  5  cc.  of  a  '5  per  cent  solution 
of  caustic  potash. 

(3)  With  a  stout  platinum  needle  carefully  scrape  the 
culture  off  the  surface  of  the  agar-agar  into  the  caustic 
potash. 

(4)  Collect  the  caustic  potash  emulsions  thus  made  in  a 
glass  mortar,  and  gently,  but  thoroughly,  rub  the  mass  up. 

(5)  Pour  the  emulsion  into  a  beaker,  and  digest  it  in  a 
water  bath  at  45°  0.,  till  it  is  liquid. 

(6)  Filter  this  through  a  small  Berkefeld  filter. 

(7)  To  the  filtrate  add  dilute  hydrochloric  acid,  as  long 
as  a  precipitate  appears. 


LESSON  ii  SEPARATION  OF  PROTEINES  145 

(8)  Filter  through  paper,  and  wash  the  residue  on  the 
filter  with  the  dilute  hydrochloric  acid,  and  then  with  dis- 
tilled water. 

(9)  Dissolve  the  washed  residue  in  the  smallest  possible 
quantity  of  a  sterile  -5  per  cent  solution  of  caustic  soda. 

Result :  alkaline  solution  of  mycoproteine. 
Tests  :  (1)  On  neutralising,  no  precipitate. 

(2)  Make  the  solution  slightly  acid,  and  add  a 
little  salt :  a  precipitate  appears. 

(b)  Bacillus  prodigiosus. 

(1)  Inoculate  twenty  potato  tubes  with  the  Bacillus  pro- 
digiosus, spreading  the  material  thoroughly  over  the  surface 
of  the  potatoes ;  and  keep  them  at  22°  C. 

(2)  When  there  is  a  copious  growth,  treat  the  cultures 
with  -5  per  cent  solution  of  caustic   potash,  as  described 
above. 

(3)  Collect  the  caustic  potash  emulsions,  and  proceed 
exactly  as  before. 

(c)  Precipitation  by  alcohol 

(1)  Inoculate  twenty  potato  tubes  with  the  Bacillus  pro- 
digiosus, and  keep  them  at  22°  0. 

(2)  When  there  is  a  copious  growth,  with  a  blunt  scalpel 
scrape  the  cultures  off  the  surface  of  the  potatoes. 

(3)  Spread  the  scrapings,  in  a  thin  layer,  over  a  plate  of 
glass,  and  exsiccate  rapidly  by  means  of  dry  heat  (up  to 
100°  C.) 

(4)  Scrape  the  dried  culture  mass  off  the  glass  plate, 

L 


146  BACTERIOLOGICAL  CHEMISTRY  LESSON  n 

and  having  placed  it  in  a  flask,  extract  it  with  distilled 
water  by  means  of  shaking. 

(5)  Filter,  and  pour  the  nitrate,  drop  by  drop,  into  an 
excess  of  absolute  alcohol :  a  precipitate  appears. 

(6)  Allow  the  precipitate  to  settle.      Separate  off  the 
alcohol,  and  dry  the  residue  at  45°  C.,  to  drive  off  the 
alcohol. 

(d)  Bacterial  extract. 

(1)  Inoculate  twenty  agar-agar  tubes  with  the  Bacillus 
pyocyaneus  (streak  cultures),  and  when  copious  growth  has 
appeared,  pour  2  to  3  cc.  of  a  sterile  -5  per  cent  caustic  potash 
solution,  or  of  distilled  water,  into  each  tube. 

(2)  Scrape  the  cultures  off  the  surface  of  the  agar-agar 
into  the  alkaline  solution  or  into  the  water. 

(3)  Collect  the  various  emulsions  in  a  flask,  and  heat  for 
ten  minutes  in  a  water  bath  at  80°  to  100°  C. 

(4)  Filter  through  a  small  Berkefeld  filter  into  a  sterilised 
flask. 


LESSON  III 

Bacterial   Colouring   Matters  —  Bacillus   Pyocyaneus  —  Bacillus 
Prodigiosus. 

Separation  of  Bacterial  Colouring  Matters  (Gessard, 
Andrewes,  Fordos) 

(a)  Bacillus  pyocyaneus  (Klein-Andrewes). 

(1)  Inoculate  twenty  agar-agar   tubes  (streak  cultures) 
from  an  actively  chromogenic  culture,  and  keep  them  at  22° 
to  30°  C. 

(2)  When  the  agar-agar  has  become  dark  green,  add  to 
each  tube  5  to  8  cc.  of  pure  chloroform,  break  up  the  agar- 
agar  with  a  glass  rod,  arid  shake  each  tube  vigorously,  till 
all  the  blue  pigment  is  dissolved  out  by  the  chloroform. 

(3)  Collect  the  chloroform  extracts  in  a  small  flask,  and 
filter  through  filter-paper  moistened  with  chloroform. 

A  clear  blue  solution  of  pyocyanine  is  thus  obtained, 
which  may  be  concentrated  by  slow  evaporation 
in  the  dark  at  38'5°  0. 

Reactions 
(1)  Evaporate  a  little  of  the  chloroform  solution  in  a 


148  BACTERIOLOGICAL  CHEMISTRY  LESSON 

porcelain  dish  at  38-5°  C.  in  the  dark  :  a  crystalline  residue 
of  pyocyanine  is  obtained. 

(2)  To  the  blue  chloroform  solution  add  dilute  sulphuric 
or  hydrochloric  acid,  drop  by  drop,  and  shake. 

"When  thoroughly  acidified,  the  solution  turns  red. 

Allow  to  settle :  the  chloroform  which  sinks  to  the 
bottom  of  the  tube  is  clear  and  colourless,  the 
supernatant  watery  solution  red. 

(3)  To  this  upper  red  layer  add,  drop  by  drop,  10  per 
cent  caustic  soda  solution  and  shake. 

The  blue  colour  reappears. 

Allow  to  settle  :  the  chloroform  which  again  sinks  to 
the  bottom  of  the  tube  takes  up  the  blue  pyo- 
cyanine, while  the  supernatant  watery  liquid 
becomes  colourless. 

(4)  Expose  the  blue  chloroform  extract  to  sunlight. 
It  soon  loses  its  blue  tint  and  becomes  yellowish. 

Now  add  caustic  soda  solution  :  the  solution  assumes 
a  violet  tint. 

(5)  Place  four  tubes,  containing  each  5  cc.  of  the  blue 
chloroform  extract,  in  boxes  behind  coloured  glass  (blue, 
green,  yellow,  red)  and  expose  them  to  the  action  of  direct 
sunlight. 

Keep  a  fifth  tube  in  the  dark  at  22°  C. 

Blue  light  discharges  the  blue  colour  rapidly,  while 
green  light  preserves  it  best. 

In  the  dark  also  it  changes  slowly. 


in  SEPARATION  OF  BACTERIAL  PIGMENTS  149 


(1)  Prepare  twenty  potato  or  agar-agar  streak  cultures 
of  the  Bacillus  prodigiosus,  which  must  be  kept  at  22°  0., 
till  the  dark  red  pigment  has  been  formed  copiously. 

(2)  To  each  tube  add  5  to  10  cc.  of  pure  ether  and  shake 
vigorously,  till  all  the  red  pigment  has  been  dissolved  out. 

(3)  Collect  the  ethereal  extracts  and  pour  them  into  a 
separating  funnel :  the  red  ethereal  extract  will  rise  to  the 
surface. 

(4)  Allow  it  to  stand  in  the  dark  for  twenty-four  hours, 
and  then  separate  the  coloured  ethereal  extract. 

(5)  Filter  this,  if  necessary,  through  paper  moistened 
with  ether. 

A  clear  red  solution  is  thus  obtained,  which  may  be 
concentrated  by  slow  evaporation  in  the  dark  at 
38-5°  C. 

Reactions 

(1)  Evaporate  a  little  in  a  porcelain  dish  at  38 '5°  C. :  a 
crystalline  residue  is  obtained. 

(2)  Add  a  few  drops  of  hydrochloric  acid  :  no  change 
in  colour. 

(3)  Add  a  few  drops  of  caustic  soda  or  caustic  potash : 
the  red   colour   is   discharged   on   shaking,  the   upper  or 
ethereal  layer  turning  yellow. 

Now  add,  drop  by  drop,  hydrochloric  acid :  the  red 
colour  will  gradually  reappear  in  the  upper  ethereal 
layer. 

(4)  Allow  the   ethereal  red  extract  to   stand  in   the 
light :  the  red  colour  disappears. 


LESSON  IV 

Peptones  and  Albumoses — Peptones — Albumoses. 

Peptones 

Tests  for  peptones. 

Prepare  a  solution  of  peptone.1 

(1)  Boil :  no  coagulation. 

(2)  Make  strongly  alkaline  with  caustic  soda,  and  add  a 
drop  of  a  dilute  solution  of  copper  sulphate  :  a  pink  colour 
is  produced  (biuret  reaction). 

(3)  Add  ammonium  sulphate  to  saturation :  no  precipitate. 

(4)  Add  nitric  acid,  drop  by  drop  :  no  precipitate. 

(5)  Add  nitric  acid  in  the  presence  of  chloride  of  sodium  : 
no  precipitate. 

(6)  Add  an  excess  of  picric  acid  :  no  precipitate. 

(7)  Add  absolute  alcohol :  a  white  precipitate,  readily 
soluble  in  water. 

(8)  Add  tannic  acid  :  a  precipitate,  soluble  in  excess. 

(9)  Add  Millon's  reagent,  and  boil :  a  red  colour. 

1  Peptone  puriss.  (Adamkiewicz),  obtained  from  E.  Merck  of  Darm- 
stadt. 


LESSON  IV 


TESTING  FOR  ALBUMOSES 


151 


Albumoses 

Tests  for  albumoses. 

Solutions  of  albumoses  are  supplied. 


(a)  Proto-albumose  (concentrated 
solution). 

(1)  Boil :  no  coagulation. 

(2)  Biuret  test :  pink  colour. 

(3)  Ammonium   sulphate :    pre- 
cipitate. 

(4)  Alcohol :  precipitate. 

(5)  Equal  volume  of  concentrated 
solution  of  sodium  chloride  in  acetic 
acid :  precipitate. 

Boil :  precipitate  disappears. 

Allow  to  cool :  precipitate  re- 
appears. 

(6)  Concentrated  aqueous    solu- 
tion of  sodium  chloride :   precipi- 
tate. 


(7)  Add  nitric  acid,  drop  by  drop, 
keeping    the    solution   cool :    pre- 
cipitate, soluble  in  excess  of  nitric 
acid. 

Warm  gently  :  precipitate  dis- 
appears. 

Cool    again :     precipitate    re- 
appears. 

(8)  Excess  of  picric  acid  :    pre- 
cipitate. 

(9)  Neutral     copper     sulphate : 
turbidity  or  precipitate. 

(10)  Boil  with  Millon's  reagent : 
red  precipitate  or  colour. 


(b)   Deutero  -  albumose    (concen- 
trated solution). 

(1)  Boil :  no  coagulation. 

(2)  Biuret  test:  pink  colour. 

(3)  Ammonium  sulphate :   pre- 
cipitate. 

(4)  Alcohol :  precipitate. 

(5)  Equal  volume  of  concentrated 
solution  of  sodium  chloride  in  acetic 
acid:  precipitate. 

Boil :  precipitate  disappears. 

Allow  to  cool :  precipitate  re- 
appears. 

(6)  Concentrated   aqueous   solu- 
tion of  sodium  chloride  :  no   pre- 
cipitate. 

Now  add  also  acetic  acid  satu- 
rated with  sodium  chloride  : 
precipitate. 

(7)  Add  nitric  acid,  drop  by  drop, 
keeping  the  solution  cool :  no  pre- 
cipitate. 

Add  a  few  crystals  of  sodium 
chloride  first  and  then  nitric 
acid,  drop  by  drop,  keeping 
the  solution  cool:  precipitate, 
which  disappears  on  warming 
and  reappears  on  cooling. 

(8)  Excess  of  picric  acid :    pre- 
cipitate. 

(9)  Neutral     copper     sulphate  : 
precipitate. 

(10)  Boil  with  Millon's  reagent : 
red  precipitate  or  colour. 


LESSON  V 

Peptones  and  Albumoses  (concluded}— Separation  of  Albumoses. 
Albumoses  (concluded) 

SEPARATION  or  DEUTERO-ALBUMOSE  FROM  PRIMARY  ALBU- 
MOSES (proto-albumose  and  hetero-albumose). 

Prepare  a  neutral  solution  of  Witte's  "peptonum  siccum"1 
in  the  least  possible  quantity  of  distilled  water. 

(1)  Add   concentrated   salt    solution:    precipitate   (I.) 
(primary  albumoses). 

(2)  Filter. 

(3)  To  filtrate  add  acetic  acid,  saturated  with  salt,  as 
long  as  a  precipitate  falls  down  :  precipitate  (II.)  (mixture 
of  proto-  with  a  little  deutero-albumose). 

(4)  Filter. 

(5)  Collect  precipitates  (I.)  and  (II.)  and  set  them  apart 
(vide  infra  (14)). 

(6)  The  filtrate,  which  contains  the  deutero-albumose,  must 
be  placed  in  a  dialysing  membrane  and  be  dialysed  against 
distilled  water,  to  remove  the  acetic  acid  and  the  salt. 

1  Obtained  from  E.  Merck  of  Darmstadt. 


LESSON  v  SEPARATION  OF  ALBUMOSES  153 

A  few  crystals  of  thymol  should  be  added,  both  to  the 
liquid  in  the  membrane  and  to  the  water  around. 

(7)  Concentrate  the  contents  of  the  membrane  in  vacuo 
at  37°  0.  to  40°  C.,  having  previously  added  a  few  thymol 
crystals. 

($)  The  concentrated  solution  should  be  poured  slowly 
into  five  to  six  times  its  volume  of  absolute  alcohol :  pre- 
cipitate (III.)  (deutero-albumose). 

(9)  Allow  the  precipitate  to  stand  under  the  alcohol  for 

several  days. 

(10)  Syphon  off  the  alcohol,  and  drive  off  the  remainder 
of  the  alcohol  by  keeping  the  precipitate  at  40°  C. 

(11)  Dissolve  the  dry  residue  in  the  smallest  possible 
quantity  of  distilled  water. 

(12)  Pour  this  solution  slowly  into  five  to  six  times  its 
volume  of  absolute  alcohol,  and  again  allow  the  precipitate 
which  appears  to  stand  under  the  alcohol  for  several  days. 

(13)  Separate  the  alcohol  as  before,  and  dry  the  pre- 
cipitate in  vacuo  at  40°  C. 

The  dry  residue  is  deutero-albumose. 

(14)  To  obtain  the  hetero-albumose  from  precipitate  (I.),  dis- 
solve the  latter  in  the  least  possible  quantity  of  distilled  water. 

(15)  Dialyse  against  running  water  for  twelve  hours,  and 
then  against  distilled  water  for  further  two  to  foui  hours,  till  all 
the  salt  has  been  removed.     (Add  thymol  crystals  as  above.) 

(16)  Pour  the  dialysed  liqufd  from  the  membrane  into  a 
beaker :  if  it  contains  a  precipitate — precipitate  (IV.) — this  is 
hetero-albumose. 

(17)  Filter  (vide  infra  (21)). 


154  BACTERIOLOGICAL  CHEMISTRY  LESSON  v 

(18)  Wash  the  residue  on  the  filter-paper  with  distilled  water, 
and  then  repeatedly  with  absolute  alcohol. 

(19)  Place  it  under  alcohol  for  several  days,  in  order  to 
dehydrate  it  completely. 

(20)  Separate  off  the  alcohol  and  dry  as  above. 
The  dry  residue  is  hetero-albumose. 

(21)  The  filtrate  from  (17)  should  be  evaporated  to  a  small 
bulk  in  vacuo  at  40°  C. 

(22)  To  the  concentrated  liquid  add  saturated  salt  solution  : 
precipitate  (V.) 

(23)  Filter. 

(24)  Dry  the  residue  at  40°  C.,  and  redissolve  it  in  the  least 
possible  quantity  of  distilled  water. 

(25)  Dialyse   the  solution,   adding  thymol  crystals,  as  de- 
scribed above. 

(26)  Concentrate  the  contents  of  the  membrane  in  vacuo  at 
40°  C. 

(27)  Pour   the    concentrated    solution    into    an    excess    of 
absolute  alcohol,  and  allow  the  precipitate  to  stand  under  the 
alcohol  for  several  days. 

(28)  Separate  off  the  alcohol,  and  dry  as  above. 
The  dry  residue  is  proto-albumose. 

Apply  the  tests  and  reactions  described  in  Lesson  IV. 
to  concentrated  solutions  of  the  albumoses. 


LESSON  VI 

Diphtheria  Albumoses — Diphtheria  Bacilli — Diphtheria  Spleen. 

Diphtheria  Albumoses  (Sidney  Martin) 

(a)  Diphtheria  bacilli. 

(1)  To  200  cc.  of  sterile  ox  serum  add  2-5  cc.  of  a  sterile 
solution  of  10  per  cent  caustic  soda  and  60  cc.  of  non- 
peptonised  sterile  broth. 

This  must  be  done  under  strictly  aseptic  conditions. 

(2)  Fill  twenty-six  tubes  with   this   solution,  using  a 
sterile  10  cc.  pipette,  and  keep  the  tubes  at  56°  C.  for  five 
to  six  days,  for  several  hours  each  day. 

(3)  Select  the  tubes  which  remain  sterile,  and  inoculate 
them  with  virulent  diphtheria  bacilli. 

(4)  Keep  them  at  38-5°  C.  for  twenty-four  to  thirty-two 
days. 

(5)  Pour  the  contents  of  tubes  into  a  large  excess  of 
strong  methylated  spirit  (1000  cc.) 

(6)  Allow  the  precipitate  to  settle,  and  to  stand  under 
the  spirit  for  a  week  or  so. 

(7)  Filter  through  paper. 


156  BACTERIOLOGICAL  CHEMISTRY  LESSON 

(8)  Extract  the  residue  with  cold  distilled  water  by 
means  of  shaking,  until  nothing  more  dissolves  out. 

(9)  Evaporate  the  watery  extract  to  a  small  bulk  at 
40°  0. 

(10)  Throw  this  concentrated  extract  into  an  excess  of 
absolute  alcohol :  precipitate  of  albumoses. 

(11)  Allow  this  to   stand  under  absolute  alcohol  for 
several  days. 

(12)  Pour  off  the  alcohol. 

(a)  Residue  =  albumoses  (b)  Alcoholic  liquid : 

Evaporate  to  dryness  at  40°  C. 
Extract    residue    several    times 

with,  absolute  alcohol : 
Deutero-albumose. 

(13)  Mix   (a)   and  (b)  together,   and   dissolve   in  the 
smallest  possible  quantity  of  distilled  water. 

(14)  Pour  the  watery  solution  into  five  to  six  times  its 
volume  of  absolute  alcohol,  and  allow  it  to  stand  for  several 
days. 

(15)  Pour  off  the  alcohol  and  dry  at  40°  C. 

(16)  Again  dissolve  in  distilled  water. 

(17)  Pour  this  solution  once   more  into  alcohol,  and 
allow  it  to  stand  for  several  days. 

(18)  Pour  off  the  alcohol,  dry,  and  redissolve  in  water, 
and  again  pour  into  absolute  alcohol. 

(19)  Allow   the   precipitate   to   stand   under   absolute 
alcohol  for  five  to  six  weeks. 

(20)  Pour  off  the  alcohol,  and  dry  the  residue  in  vacuo 
over  sulphuric  acid. 


vi  DIPHTHERIA  ALBUMOSES  157 

Result :  a  yellowish  brown  powder,  consisting  chiefly 

of  deutero-albumose : 

(a)  insoluble  in  chloroform,  ether,  alcohol,  am- 
monium sulphate,  and  nitric  acid,  in  the 
presence  of  sodium  chloride ; 

(1)  soluble  in  cold  or  boiling  water ;  • 
(c)  giving  a  marked  biuret  reaction. 

For  the  separation  of  the  albumoses  see  page  152. 

(b)  Diphtheria  spleen. 

(1)  Mince  the  spleen  of  a  child  dead  of  diphtheria. 

(2)  Throw  the  finely  minced  mass  into  a  large  excess  of 
strong  methylated  spirit,  and  allow  it  to  stand  for  several 
weeks. 

(3)  Separate  the  spirit  by  means  of  filtering. 

(4)  Then  proceed  exactly  as  described  above  (see  (a)). 
Albumoses,  especially  deutero-albumose,  will  be  obtained. 


LESSON  VII 

Diphtheria  Toxine — Action  of  Magnesium  Sulphate  and  Ammonium 
Sulphate  on  Sulphate  of  Quinine. 

Diphtheria  Toxine  (Uschinsky) 

(1)  PREPARE  a  solution  of  aspartate  of  sodium : 

Water     .                     .  .  1000  cc. 

Glycerine          .          .  .  35  cc. 

Sodium  chloride          .  .  6  grammes. 

Calcium  chloride          .  .  '1  gramme. 

Magnesium  sulphate    .  .  '2-*4  grammes. 

Bi-potassium  phosphate  .  2-2-5  grammes. 

Lactate  of  ammonium .  .  6-7  cc. 

Sodium  aspartate         .  .  3 '4  grammes. 

(2)  Mix  thoroughly,  and  heat  for  half  an  hour  at  about 
40°  0. 

(3)  Neutralise  with  sodium  carbonate,  and  again  heat 
for  half  an  hour. 

(4)  Filter. 

(5)  Pour  the  filtrate  into  flasks,  and  sterilise  in  the 
steamer  in  the  ordinary  manner. 


LESSON  vii  TOXINE  REACTIONS  159 

(6)  Inoculate  two  flasks,  containing  100  cc.  each,  with 
Bacillus  diphtherise. 

(7)  After  twenty-four  to  thirty-two  days  filter  the  cul- 
tures through  a  Berkefeld  filter. 

(8)  Evaporate  the^filtrate  down  to  a  small  bulk  at  40°  C. 

Reactions 

(a)  Millon's  reaction  :  more  or  less  typical. 

(b)  Xanthoproteic  reaction  :  more  or  less  typical. 

(c)  Alcohol :  precipitate. 

(d)  Acetic  acid  and  ferrocyanide  of  potassium :  tur- 

bidity after  some  time. 

(e)  Phospho-molybdic  acid  :  slight  precipitate. 

(/)  Ammonium  sulphate :  no  precipitate ;  hence  no 
albumoses  present. 

(g)    No    biuret    reaction;    hence    no  albumoses   or 
peptones  present. 

To  obtain  the  toxine  in  a  dry  condition,  pour  the  filtrate 
into  an  excess  absolute  alcohol  after  it  has  been  evaporated 
down  to  a  small  bulk. 

Allow  the  precipitate  to  settle  and  to  stand  under 
alcohol  for  a  few  days. 

Separate  off  the  alcohol,  and  dry  the  residue  at  40°  0. 

The  readiest  method,  for  all  practical  purposes,  of 
obtaining  diphtheria  toxine  in  solution  is  to  filter 
virulent  broth  cultures  through  a  Berkefeld  filter, 
as  described  on  p.  141. 


160  BACTERIOLOGICAL  CHEMISTRY         LESSON  vil 

Action  of  Magnesium  Sulphate  or  Ammonium  Sulphate 
on  Sulphate  of  Quinine  (Duclaux) 

(a)  Prepare  a  cold  and  almost  saturated  solution  of 
sulphate  of  quinine. 

To  it  add  10  per  cent  of  finely  powdered  magnesium 
sulphate. 

A  precipitate  appears. 

(b)  Dilute  a  cold  and  almost  saturated  solution  of  sul- 
phate of  quinine  with  an  equal  volume  of  water. 

Then  add  finely  powered  magnesium  sulphate. 

A  precipitate  does  not  appear  until  30  per  cent  of 
the  salt  has  been  added. 

(c)  Prepare  a  cold  and  saturated  solution  of  sulphate  of 
quinine,  and  dilute  it  with  one-tenth  its  volume  of  water. 

(1)  Gradually  add  finely  powdered  ammonium  sul- 
phate, until  no  more  precipitate  appears. 

Filter. 

(2)  To  the  filtrate  again  add  ammonium  sulphate, 
until  no  more  precipitate  appears. 

Filter. 

(3)  To  the  filtrate  again  add  ammonium  sulphate, 
until  no  more  precipitate  appears. 

The  appearance  of  a  precipitate,  therefore,  depends 
greatly  on  the  degree  of  concentration. 


LESSON  VIII 

Ferments  and  Enzymes— Action  of  Chloroform  on  Ferments  and 
Enzymes — Action  of  Heat  on  Enzymes— Separation  of  Enzymes 
(Alcohol  Precipitation). 


Ferments  and  Enzymes 
A.  FERMENT  AND  ENZYME  IN  YEAST. 

(1)  Prepare  a  dilute  solution  of  cane  sugar. 

.  (2)  Test  it  with  Fehling's   solution  :    no  reduction  of 
copper. 

(3)  Pour  a  little  of  this  sugar  solution  into  two  test-tubes, 
and  add  a  little  yeast. 

(4)  Keep  both  tubes  at  38 -5°  C. 

(5)  After  a  few  hours  (four  to  six  hours)  test  one  tube 
with  Fehling's  solution  :  marked  reduction  of  copper. 

Invertine   splits   up   cane   sugar   into  dextrose  and 
Isevulose. 

(6)  Test  the  other  tube  next  day  with  Fehling's  solution : 
no  reduction  of  copper. 

Alcohol  fermentation  has  taken  place. 
M 


162  BACTERIOLOGICAL  CHEMISTRY  LESSON 

B.  ACTION    OF    CHLOROFORM    ON    FERMENTS    AND 
ENZYMES. 

(1)  Suspend  a  little  yeast  in  lukewarm  sterile  water. 

(2)  Pour  a  little  of  this  suspension  into  two  large  test- 
tubes. 

(3)  Shake  up  the  contents  of  one  of  these  tubes  with  an 
equal  volume  of  chloroform  for  five  to  six  minutes,  but 
leave  the  other  for  control  purposes  (vide  infra). 

(4)  Allow  the  chloroform  to  settle  to  the  bottom  of  the 
tube. 

(5)  Add  a  little  of  the  supernatant  liquid  to  two  test- 
tubes,  containing  a  dilute  solution  of  cane  sugar. 

(6)  Keep  these  tubes  at  38 -5°  C. 

Test  one  tube  with  Fehling's  solution  after  four  to 
six  hours  :  marked  reduction  of  copper. 

Test  the   other  tube  with  Fehling's  solution  after 
twenty-four  hours  :  marked  reduction  of  copper. 

(7)  The  other  tube,   containing  a   little  of  the  yeast 
suspension,  must  not  be  shaken  up  with  chloroform. 

(8)  Add  a  little  of  the  original  suspension  to  two  tubes, 
containing  a  dilute  solution  of  cane  sugar. 

(9)  Keep  these  tubes  at  38 -5°  C. 

Test  one  tube  with  Fehling's  solution  after  four  to 
six  hours :  marked  reduction  of  copper. 

Test  the  other  tube  with  Fehling's   solution  after 
twenty-four  hours  :  no  copper  reduction. 


vin  ENZYMES  163 

Conclusions  : 

Invertine  is  an  enzyme,  and  therefore  not  destroyed 
by  chloroform. 

The  alcohol -producing  ferment  is  a  living  ferment, 
and  as  such  is  destroyed  by  chloroform. 

C.  ACTION  OF  MOIST  HEAT  ON  ENZYMES. 

(1)  Prepare  a  suspension  of  yeast  in  lukewarm  sterile 
water. 

(2)  Shake  up  a  little  of  it  with  chloroform  as  above,  and 
allow  the  chloroform  to  settle. 

(3)  Separate  the  supernatant  liquid,  and  pour  a  little  of 
it  into  two  test-tubes. 

(4)  Heat  one  of  them  up  to  boiling  point,  leaving  the 
other  for  control. 

(5)  Add  some  of  the  boiled  suspension  to  two  tubes  con- 
taining a  dilute  solution  of  cane  sugar,  and  keep  the  tubes 
at  38-5°  C. 

Test  one  tube  with  Fehling's  solution  after  four  to 
six  hours  :  no  reduction  of  copper  and  no  alcohol. 

Test  the  other  tube  with  Fehling's  solution  after 
twenty-four  hours  :  no  reduction  of  copper  and  no 
alcohol. 

(6)  Add  some  of  the  suspension  in  the  control  tube  to 
two  tubes  containing  a  dilute  solution  of  cane  sugar,  and 
keep  the  tubes  at  38 -5°  C. 

Test  one  tube  with  Fehling's  solution  after  four  to 
six  hours  :  marked  reduction  of  copper. 


164  BACTERIOLOGICAL  CHEMISTRY  LESSON 

Test  the  other  tube  with  Fehling's   solution   after 
twenty-four  hours  :  marked  reduction  of  copper. 

Boiling  destroys  the  enzymes  in  solution. 

D.  SEPARATION  or  ENZYMES. 

(a)  Precipitation  by  alcohol  (Barth). 

(1)  Fresh  yeast  is  dried  at  the  ordinary  temperature  in 
vacuo  over  sulphuric  acid  (a  fairly  large  quantity  of  yeast 
must  be  used). 

(2)  Rub  up  the  dried  yeast  into  a  fine  powder. 

(3)  Dry  the  powder  in  the  hot-air  chamber  at  100°  C. 
for  six  hours. 

(4)  Allow  it  to  cool,  and  when  it  is  quite  cold  add 
distilled  water,  so  as  to  convert  it  into  a  thin  mess. 

(5)  Let  this  suspension  stand  and  settle  at  40°  C.  for 
twelve  hours. 

(6)  Decant  the  supernatant  water,  and  filter  it  till  it  is 
clear. 

(7)  Pour  the  filtrate  into  five  to  six  times  its  volume  of 
90  per  cent  spirit :  a  precipitate  appears. 

(8)  Allow  the  precipitate  to  settle,  and  filter  at  once. 

(9)  Wash  the  residue  on  the  filter-paper  with  absolute 
alcohol. 

(10)  Eemove  the  alcohol  by  means  of   pressure,   and 
shake  the  residue  up  with  water. 

(11)  Again   filter,   and   precipitate    the    filtrate   with 
alcohol. 


vni  TESTS  FOR  ENZYMES  165 

(12)  Filter,  and  wash  the  residue  on  the  paper  with 
absolute  alcohol  as  before. 

(13)  Remove   the   alcohol  by  means  of   pressure   and 
by  drying  at  40°  C. 

(14)  Complete  the  drying  in  mew  over  sulphuric  acid. 
The  resulting  powder  is  invertine. 

Instead  of  using  dried  yeast,  a  suspension  of  yeast  in 
lukewarm  water  may  be  shaken  up  with  an  equal  volume 
of  chloroform  in  a  shaking  machine  for  twenty  to  thirty 
minutes. 

The  chloroform  should  be  allowed  to  settle,  and  the 
supernatant  watery  suspension  poured  into  three  times  its 
volume  of  90  per  cent  spirit. 

The  resulting  precipitate  must  be  treated  as  described 
above  under  (8)  to  (14). 

Tests 

(1)  Add  a  little  of  the  powder  to  two  test-tubes  con- 
taining a  dilute  solution  of  cane  sugar,  and  keep  the  tubes 
at  38-5°  C. 

Test  one  tube  with  Fehling's  solution  after  four  to 
six  hours  :  reduction  of  copper,  no  alcohol. 

Test  the  other  tube  with   Fehling's  solution   after 
twenty-four  hours  :  reduction  of  copper,  no  alcohol. 

(2)  Heat  some  of  the  dry  powder  to  100°  C.  for  a  few 
minutes,   and  then  add  a  little  of  it  to  two   test-tubes 
containing  a  dilute  solution  of  cane  sugar.     Keep  the  tubes 
at  38-5°. 


166  BACTERIOLOGICAL  CHEMISTRY        LESSON  vm 

Test  them  as  before  with  Fehling's  solution,  after 
four  to  six  hours,  and  again  after  twenty-four 
hours  :  reduction  of  copper,  no  alcohol. 

(3)  Heat  some  of  the  dry  powder  at  130°  or  135°  C.  for 
fifteen  minutes,  and  repeat  the  previous  experiment. 

On  testing  with  Fehling's  solution,  no  reduction  of 
copper  or  alcohol. 

Conclusions  : 

Enzymes  in  solution  are  readily  rendered  inert  by 

heat. 
Enzymes  in  a  solid  and  dry  condition  are  destroyed 

with  difficulty  by  heat. 


LESSON  IX 

Ferments  and  Enzymes  (concluded}— Precipitation  by  Calcium  Phosphate 
— Proteolytic  Enzymes. 

Ferments  and  Enzymes  (concluded] 

D.  SEPARATION  OF  ENZYMES  (concluded). 

(b)  Precipitation  by  calcium  phosphate  (Von  Briicke). 

(1)  Prepare  a  suspension  of  fresh  yeast  in  dilute  phos- 
phoric acid  (a  fairly  large  quantity  of  yeast  must  be  used). 

(2)  Keep  this  suspension  at  38'5°  0.  for  five  to  six  days. 

(3)  Now  neutralise  it  with  lime-water:    precipitate  of 
calcium  phosphate,  which  carries  the  enzyme  down  with  it. 

(4)  Filter,  and  thoroughly  wash  the  precipitate  with 
distilled  water. 

(5)  Dissolve  the  precipitate  in  the  least  possible  amount 
of  water,  acidulated  with  hydrochloric  acid. 

(6)  Dialyse  the   resulting  acid  solution,  replacing  the 
hydrochloric  acid  from  time  to  time,  so  as  to  keep  the 
solution  within  the  membrane  acid. 

(7)  When  all  the  calcium  phosphate,  and  eventually  the 
hydrochloric  acid,  have  been  removed  by  dialysis,  pour  the 


168  BACTERIOLOGICAL  CHEMISTRY  LESSON 

contents  of  the  membrane,  slowly  and  gradually,  into  an 
excess  of  alcohol :  a  precipitate  appears. 

(8)  Filter  at  once,  and  collect  the  residue. 

(9)  Dry  the  latter,  and  remove  the  alcohol  by  means  of 
heat  at  40°  C.,   and  complete  the  drying  in  vacuo  over 
sulphuric  acid. 

Test  the   resulting   powder  (invertine)  as  described 
above  (vide  p.  165). 

E.  PROTEOLYTIC  (TRYPTIC)  ENZYMES  OF  MICRO-ORGAN- 
ISMS  WHICH   LIQUEFY  GELATINE   (Fermi). 

(1)  Prepare  thirty  roll  tubes  severally  of 

(a)  Bacillus  prodigiosus ; 

(b)  Bacillus  pyocyaneus ; 

(c)  Vibrio  Finkler-Prior ; 

(d)  Vibrio  cholerse  Asiaticse. 

(2)  When  the  gelatine  is  liquefied  collect  the  contents 
of  the  tubes  in  small  flasks. 

(3)  To  200  cc.  of  liquefied  gelatine  culture  add  200  cc. 
of  dilute  alcohol,  viz.  : 

(a)  in  the  case  of  the  Bacillus  prodigiosus,  65  per  cent 

alcohol ; 

(b)  in  the  case  of  the  Bacillus  pyocyaneus,  75  per  cent 

alcohol ; 

(c)  in  the  case  of  the  Vibrio  Finkler-Prior,  70  per  cent 

alcohol ; 

(d)  in  the  case  of  the  Vibrio  choleras  Asiaticse,  65 

per  cent  alcohol. 


rx  TRYPTIC  ENZYMES  169 

(4)  Allow  to  stand  for  twenty-four  hours,  and  then  filter 
through  paper. 

(5)  To  each  of  the  four  filtrates  add  absolute  alcohol  in 

excess  :  a  precipitate  appears. 

(6)  Filter,  and  dry  the  four  precipitates  at  40°  C.  in  the 
ordinary  manner. 

(7)  Dissolve  the  dry  residues  severally  in   100  cc.  of 
saturated  thymol  water  :  enzyme  solutions. 

Tests  for  tryptic  enzymes 

(1)  To  four  tubes  of   thymol  gelatine  (7  grammes  of 
gelatine  and  100  cc.  of  thymol  water)  severally  add  5  cc. 
of  the  ferment  solutions. 

Allow  the  tubes  to  stand  at  15°  C. 

The  gelatine  will  be  liquefied  after  a  time. 

(2)  Place  "5  gramme  of  dried  fibrin  in  two  test-tubes. 

Add  to  one  of  them  5  cc.  of  the  enzyme  solution  of 
the  Bacillus  prodigiosus,  and  to  the  other  5  cc.  of 
the  enzyme  solution  of  the  Vibrio  Finkler-Prior. 

Examine  the  tubes  after  eight  hours :  the  fibrin  is 
almost  entirely  liquefied  in  the  case  of  the  former 
enzyme,  and  partially  so  in  the  case  of  the  latter. 

F.  TRYPTIC  ENZYMES  OF  BACILLUS  PRODIGIOSUS  OR 
BACILLUS  PYOCYANEUS  (Fermi). 

(1)  Grow    the    Bacillus   prodigiosus    or    the    Bacillus 
pyocyaneus  in  the  following  solution  : 


170  BACTERIOLOGICAL  CHEMISTRY  LESSON 

Ammonium  phosphate  .  10  grammes. 

Bi-potassium  phosphate  .  1  gramme. 

Magnesium  sulphate     .  •  '2  gramme. 

Glycerine.           .           .  .  40-50  cc. 

Distilled  water  .           .  .  1000  cc. 

(2)  Two  to  three  litres  should  be  prepared   and  dis- 
tributed in  twenty  to  thirty  small  flasks. 

(3)  Allow  the  inoculated  flasks  to  stand  at  30°  C.  for  a 
week. 

(4)  Filter  the  cultures  through  a  Berkefeld  filter. 

(5)  Evaporate  the  filtrate  to  a  small  bulk  in  a  vacuum 
pan. 

(6)  Pour  this  concentrated  solution  into  eight  to  ten 
times  its  volume  of  absolute  alcohol :  a  precipitate  appears. 

(7)  Filter  through  a  paper  filter,  and  thoroughly  wash 
the  residue  on  the  paper  with  absolute  alcohol. 

(8)  Redissolve  the  washed  residue  in  the  least  possible 
quantity  of  distilled  water. 

(9)  Dialyse   against   distilled  water   or   running  water 
for  twelve  to  twenty-four  hours. 

(10)  Now  evaporate  the  dialysed  solution  to  a  small 
bulk  in  a  vacuum  pan. 

(11)  Pour  the  concentrated  solution  into  eight  to  ten 
times  its  volume  of  absolute  alcohol  as  before. 

(12)  Filter  through  a  paper  filter,  and  wash  the  residue 
with  absolute  alcohol. 

(13)  Collect  the  washed  residue  in  a  small  glass  dish, 
and  drive  off  the  alcohol  by  heating  it  to  45°  C. 


ix  TESTING  OF  TRYPTIC  ACTION  171 

To  test  the  tryptic  action  of  the  dry  powder,  dissolve 
a  little  of  it  in  a  small  quantity  of  a  '5  to  1  per 
cent  sterilised  carbolic  acid  solution  and  add  it  to 
a  tube  of  carbol  gelatine  (5  to  7  grammes  of  gelatine 
to  100  cc.  of  a  1  to  2  per  cent  solution  of  carbolic 
acid). 

The  gelatine  will  be  liquefied. 


LESSON  X 

Ptomaines — Cadaverine — Putrescine 

Ptomaines 
CADAVERINE  AND  PUTRESCINE  (Udransky  and  Baumann). 

(1)  Allow  a  solution   of  white  of   egg  to  putrefy  for 
several  days. 

(2)  Distil  to  a  small  volume :  indol,  scatol,  and  phenol 
pass  over  in  the  distillate,  and  are  disregarded. 

(3)  Filter  the  residue. 

(4)  To  the  filtrate  add  an  equal  volume  of  a   10  per 
cent  solution  of  caustic  soda. 

(5)  Shake,  and  gradually  add,  drop  by  drop,  a  solution  of 
benzoyl  chloride  :  a  crystalline  precipitate  appears. 

(6)  Allow  to  stand  for  several  days. 

(7)  Filter. 

(a)  Strongly  acidulate  the  turbid  (a)  The  residue  must  be  digested 

filtrate  with  sulphuric  acid  (benzoic       in  spirit, 
acid  passes  off). 

(6)  Shake  up  with  three  times  (6)  Filter    and    evaporate    the 

its  volume  of  ether  and  filtrate  to  a  small  bulk. 


LESSON  X 


SEPARATION  OF  PTOMAINES 


173 


(c)  Separate  the  ethereal  extract. 

(d)  Kepeat  this  three  times  and 
collect  the  ethereal  extracts. 

(e)  Distil  off  the  ether. 

(/)  Neutralise  the  residue,  before 
it  sets,  with  12  per  cent  caustic 
soda  :  turbid  liquid. 

(g)  Mix  with  three  to  four  times 
its  volume  of  12  per  cent  caustic 
soda,  and  keep  in  the  cold  for 
twelve  to  twenty -four  hours. 

(h)  Eemove  the  liquid  from  the 
crystals  which  have  formed  and 
wash  the  crystalline  residue  with 
cold  caustic  soda. 

(i)  Now  wash  thoroughly  with 
water. 

(k)  Dissolve  the  crystals  in  warm 
spirit. 


(1}  Add  a  large  excess  of  water ; 
the  crystals  are  reprecipitated. 

(m)  Allow  to  settle  and  filter. 


(c)  Pour  this  into  thirty  times 
its  volume  of  cold  water. 

(d)  Allow  to  stand  for  several 
days  :  crystals  appear. 

(e)  Filter. 

(/)  Wash  crystalline  residue  with 
water,  until  the  washings  are  quite 
clear. 

(g)  Press  out  as  much  of  the 
water  as  possible. 


(h)  Dissolve  the  crystalline  resi- 
due in  the  least  possible  quantity 
of  absolute  alcohol. 

(i)  Pour  this  solution  into  a 
large  excess  of  water  :  precipitate. 

(k)  Filter  again  and  redissolve 
the  residue  in  the  least  possible 
quantity  of  warm  alcohol :  crystal- 
line precipitate  will  appear. 


(8)  Mix  the  two  crystalline  precipitates,  and  dissolve 
them  in  the  least  possible  quantity  of  warm  alcohol. 

(9)  Pour  the  alcoholic  solution  into  twenty  times  its 
volume  of  ether. 

(10)  Shake,  and  allow  to  crystallise. 

(11)  Filter:  residue  consists  of  the  benzoyl  compound 
of  tetramethylene-diamine  or  putrescine. 

(12)  Filtrate  :  distil  off  the  alcohol  and  ether. 

(13)  Crystals    appear:    benzoyl    compound    of   pentcir 
methylene  diamine  or  cadaverine. 


174 


BACTERIOLOGICAL  CHEMISTRY 


LESSON  X 


(14)  The  crystals  of  these  two  substances  may  be  purified 
by  dissolving  them  in  spirit  and  allowing  them  to  crystal- 
lise out  again. 


I.  Tetramethylene-diamine 
(putrescine). 

(a)  Dissolve  crystals  in  a  solu- 
tion of  equal  volumes  of  alcohol 
and  concentrated  hydrochloric  acid. 

(6)  Heat  in  water  bath  at  45°  C. 
for  twelve  hours. 

(c)  Dilute  with   water,   till    no 
more  precipitate  appears. 

(d)  Filter. 

(e)  Filtrate   shake   with    ether, 
and  separate  the  ethereal  extract. 

(/)  Slowly  evaporate  the  ethereal 
extract :  crystalline  mass,  with 
difficulty  soluble  in  alcohol  = 
hydrochlorate  of  putrescine. 

(g)  To  a  concentrated  aqueous 
solution  add  an  alcoholic  solution 
of  platinum  chloride :  crystalline 
double  salt. 

(h)  Dissolve  in  hot  water  and 
recrystallise. 


II.  Pentamethylene-diamine 
(cadaverine). 

(a)  Dissolve  crystals  in  a  solu- 
tion of  equal  volumes  of  alcohol 
and  concentrated  hydrochloric  acid. 

(6)  Heat  in  water  bath  at  45°  C. 
for  two  days. 

(c)  Dilute   with   water,  till  no 
more  precipitate  appears. 

(d)  Filter. 

(e)  Filtrate   shake  with    ether, 
and  separate  the  ethereal  extract. 

(/)  Slowly  evaporate  the  ethereal 
extract :  crystalline  mass,  soluble 
in  water,  not  readily  soluble  in 
alcohol  =  hydrochlorate  of  cada- 
verine. 

(g)  To  a  concentrated  spirit 
solution  add  an  alcoholic  solution 
of  platinum  chloride :  crystalline 
double  salt. 

(h)  Dissolve  in  hot  water  and 
recrystallise. 


Cultures  of  cholera  and  Finkler-Prior  vibrios  may  be 
examined  for  ptomaines  in  the  same  manner. 


INDEX 


ACETIC  acid  for  clearing,  9 

decolourising,  10 
Acid  alcohol  for  decolourising,  30, 

37 

Actinomyces,  45,  46,  69,  70 
Actinomycosis,  45,  46,  69,  70 

paraffin  sections,  69 

staining  of  mycelium,  69 
clubs  and  mycelium,  70 
Agar-agar,   preparation   of  nutri- 
ent, 90 

plates,  103,  104 

sterilisation  of,  91 

to  fill  tubes  with,  90 
Air,  examination  of,  108-115 

anaerobic  germs,  113-115 
Albumoses,  151-155 

diphtheria,  155-157 

separation  of,  152 

tests  for,  151 
Ammonia,   disinfectant   action   of, 

131 
Anaerobic  organisms,  113-119 

in  air  and  dust,  113-115 

in  soil,  115-119 

cultivation  of,  113-118 

separation  of,  118,  119 
Anaerobic  putrefaction,  120,  121 
Andrewes,  147 
Aniline  dyes,  9 

fuchsine,  30 

gentian  violet,  18 

water,  18 

xylol,  52 


Anthrax   bacillus,  21-30,  32,  34- 
37,  71-77 

asporogenous  cultivation,  22 

attenuation,  23,  25,  26 

cultivation  of,  21 

examination  of  aniinaldead  of,  132 

frozen  sections,  35 

gelatine  plates  of,  29,  34 

hanging  drop,  22,  24,  25 

immunity  of  frog,  73,  74 

impression  specimens,  26',  34 

in  tissues,  27,  35 

litmus  agar-agar,  25,  26,  29 

phagocytosis,  71-77 

production  of  acid  by,  22 

silk  threads,  127 

staining  of,  23,  24 
spores  of,  30,  32 

Antiseptics,  method  of  testing  (car- 
bolic acid),  126 
Aspartate  of  sodium,  158 
Aspergillus  niger,  cultivation  of,  6 

examination  of,  13 
Aspirator,  110,  111 
Avian  tubercle,  67 

BACILLUS  anthracis  (vide  Anthrax) 
coli   communis  (vide  Bacterium 

coli  commune) 
diphtherias  (vide  Diphtheria) 
filamentosus,  8,  14,  17,  32 
fluorescens,  6,  21,  97 
hay,  33 
leprse  (vide  Leprosy) 


176 


INDEX 


Bacillus  mallei,  68,  69 

megatherium,  32 

of  glanders,  68,  69 

of  tetanus,  69,  116-118 

of  typhoid  fever  (vide  Typhoid) 

prodigiosus,  5,  12,  21,  126, 136- 
138,  142,  145,  149,  168- 
170 

pyocyaneus,  4,  132,  141,  142, 
144,  146,  147,  168-170 

subtilis,  33 

tuberculosis  (vide  Tubercle) 

typhosus  (vide  Typhoid) 
Bacterial  colouring  matter,  147 

enzymes,  167 

extracts,  146 

ferments,  167 

metabolic  products,  141 

poisons,  141 

products,  141 

toxines,    141    (vide    Albumoses, 

Ptomaines,  Protei'nes) 
Bacterium  coli  commune,  21,  53- 
55,  59,  98-100,  106,  120, 
124 

cultivation  of,  53 

curdling  ferment,  21,  55 

gas  formation  by,  54 

in  ice  cream,  124,  125 

in  meat,  120,  121 

in  milk,  106 

in  water,  98-100 

shake  cultures,  54 

staining  of,  54 

varieties  of,  54 
Baumann,  172 
Beakers,  cleaning  of,  82 

sterilisation  of,  83 
Beef  broth,  preparation  of,  83 

sterilisation  of,  85 

to  fill  tubes  with,  84 
Berkefeld  filter  (vide  Filter) 
Blood  serum,  153 

(Lorrain  Smith),  preparation  of, 
92 

sterilisation  of,  92 
Broth  (vide  Beef  broth) 
Briicke,  Von,  167 


CADAVERINE,  172-174 

hydrochlorate  of,  174 
Calcium   phosphate  and  fi 

167 

Carbol  fuchsine,  30 
Carbol  gelatine,  88,  171 
Carbol  gentian-violet,  61 
Carbolised  sputum,  pneumonic,  56 

tubercular,  61 
Celloidin  sections,  45,  46 
Chemiotaxis,  77 
Chlorine,    disinfectant    action    of, 

131 

Chloroform,    action   on   invertine. 
162 

enzymes,  162 

ferments,  162 
Cholera,  37-45 

action  of  sunlight  on,  43 

cultivations,  37 

enzymes,  168 

examination  of  animal  dead  of, 
41,  42,  132 

examination  of  water,  101-104 

flagella,  38,  39 

hanging  drop  cultures,  41,  45 

nitrous  acid  in  cultures,  143 

plates,  42,  44 

red,  143 

staining  of  vibrio,  40 

varieties,  44,  45 

Chromogenic    organisms,    3,     147 
(vide    Bacillus    prodigiosus 
and  Bacillus  pyocyaneus) 
Cladothrix  asteroides,  45 

nivea,  45 
Cleaning  of  beakers,  82 

flasks,  82 

new  test-tubes,  81 

used  test-tubes,  82 
Clearing  of  films  with  acetic  acid, 
9 

tissues,  35 

frozen  sections,  35 

with  aniline  xylol,  52 
Colonies,  counting  of,  95,  96 
Colouring  matters,  bacterial,  147 
Counting  of  colonies,  95,  96 


INDEX 


177 


ass  specimens  (vide  Films) 

ag  of,  38 

.ce,  124.  125 

,ions,  agar-agar,  3,  7 

Q 

ge.   .ine,  5,  7 

po1.  ito,  6 

in  hanging  drops,  14,  17 

in  plates,  29,  103 
Curdling  ferment,  21,  45 
Cysticercus,  123 
Czinzinski's  solution,  35 

DEUTERO  -  ALBUMOSE,     1 52     (vide 

Albumoses) 

Diphtheria  albumoses,  155-157 
separation  from  cultures,  155, 

157 

separation  from  spleen,  157 
cultivations,  57 
films,  59 
hanging  drop,  57 
in  frozen  sections,  57 
in  milk,  106 
in  paraffin,  65,  66 
membrane,  65,  66,  78 
staining  of,  57 
toxines,  158 

Disinfectant  action  of  gases,  129-131 
sulphur  dioxide,  129 
ammonia,  131 
chlorine,  131 
Disinfectants,    method    of  testing, 

127-131 

Koch's  method,  127 
Sternberg's  method,  128 
Distilled  water,  examination  of,  96 
Double   staining,   with  cosine  and 
methylene-blue,  28,  36,  72, 
73,75 

with  Czinzinski's  fluid,  35 
with  methylene-blue  and  picro- 

carmine,  36 

(vide  also  Gram's  method) 
Duclaux,  160 
Dust  (vide  Air) 

EMBEDDING  in  paraffin,  133,  135 


Endocarditis,  ulcerative,  57 
Enzymes,  161-171 

action  of  chloroform  on,  162 

action  of  moist  heat  on,  1 63 

precipitation  by   calcium   phos- 
phate, 167 

proteolytic,  168 

separation  of,  164 

tests  for,  165 

tryptic,  169-171 
Eosine,  28 

as  counterstain,  20 

as  double  stain,  35 
Eosinophile  granules,  28 

leucocytes,  28,  73-77 
Ermengem,  Van,  38 
Erysipelas,  51 

Examination  of  air,  109-115  (vide 
Air) 

anaerobic  germs,  113-115 

animal  (anthrax),  27 
(cholera),  44 

dead   of  a  bacterial    disease 
132-135 

antiseptics,  126 

decomposing  meat,  120-124 

diseased  meat,  120-124 

disinfectant  gases,  129-131 

disinfectant  solutions,  127 

dust  (vide  Air) 

filters  (vide  Filters) 

fresh  tissues  (films),  27,  28 

frozen  sections,  35 

gelatine  plates,  34,  44 

ice  cream,  124,  125 

milk,  105-108 

soil,  115,  118 

stained  films,  9-10 

tubercular  sputum,  59-61 

water,  93-104 

FERMENT,  161-166  (vide  Enzyme) 
action  of  chloroform  on,  162 
bacterial,  169 
curdling  ,  21,  45 

Fermi,  168,  169 

Fibrin,  staining  of,  51,  52 

Films,  preparation  of,  8,  19 


178 


INDEX 


Films,  preparation  of,  from  anthrax 

tissues,  27,  28 

Filter,    Berkefeld,    98,    106,    125, 
136,  141,  142 

effect  of  use  on,  137 

testing  of,  136 

sugar,  110-112 

Flagella,  staining  of,  38,  39,  46 
Flasks,  cleaning  of,  82 

sterilisation  of,  83 
Fordos,  147 
Frozen  sections,  35,  50-52,  62,  63 

anthrax,  35 

tubercle,  62,  63 
Fuchsine,  aqueous  solution,  9 

aniline,  30 

carbol,  31 

Ehrlich's,  30 

Loffler's,  30 

Ziehl's,  31 

GELATINE,  carbol,  171 

thymol,  169  * 

plates,  examination  of,  34 

preparation  of  nutrient,  86 

sterilisation  of,  87 

to  fill  tubes  with,  87 
Gentian- violet,  aqueous  solution,  9 

aniline  water,  18 

carbol,  61 
Gessard,  147 

Glanders,   staining  of  paraffin  sec- 
tions, 68,  69 
Glycerine  broth,  85 

agar-agar,  preparation  of,  91 
Gonorrhoea,  48 
Gram's  iodine  solution,  18 
Gram's   method  of    staining,    an- 
thrax, 27 

celloidin  sections,  45,  46 

films,  17,  18,  19 

frozen  sections,  36,  37 

paraffin  sections,  70,  71 

pus,  20 

tubercle,  63 

Grape-sugar  agar-agar,  preparation 
of,  91 

to  fill  tubes  with,  91 


Grape-sugar  broth,  preparation  of, 

85 

gelatine,  preparation  of,  87 
Griess's  reaction,  143 
Gruber's  method,  103 

HANGING  drop  cultures,  14,  17 

examination  of,  15 

staining  of,  17-19 
phagocytosis  in,  71,  72,  76,  77 
Hardening  of  tissues,  133,  134 
Hetero-albumoses,  152,  153,  154 

ICE  cream,  examination  of,  124,  125 
Immunity,    experiments    on    IVog, 

73,  74 
Impression  specimens — 

gelatine  plates,  34 

sloped  gelatine,  26 

staining  of,  26 
Indol  reaction,  100 
"  Intracellular  "  poisons,  142 
Invertine,  161-168 

action  of  chloroform  on,  162 

separation  of,  164 
Iodine,  Gram's  solution,  18 

Weigert's  solution,  52 

KLEIN,  147 

LEPROSY  bacillus,  57 
Leucocytes,  staining  of,  28 

eosinophile,  73-77 
Loffler's  methylene-blue,  12,  13 

MADURA  disease,  46 

Malignant  oedema,  116-118 

Mammalian  tubercle,  67 

Martin,  155 

Meat  infusion,  preparation  of,  86 

Metabolic  products  of  bacteria,  141 
(vide  Toxines,  Albumoses, 
Prote'ines,  Ptomaines)  • 

Method,  Gram's,  17 
Gruber's  (cholera),  103 
Pitfield's  (flagella),  46 
Van  Ermengem's  (flagella),  38 


INDEX 


179 


Method,  Van  Ketel's,  61,  107 

Weigert's,  51,  52,  66,  69 
Methylene-blue,  aqueous  solution,  9 
Loffler's,  12,  13 
and  picrocarmine,  36 
Micrococci :  pueumoniae  (see  Pneu- 
monia) 

pyogenic,  50-52 
Staphylococcus  cereus  flavus,  7, 

47,  48 
Staphylococcus  pyogenes   albus, 

47,  48 
Staphylococcus  pyogenes  aureus, 

7,  8,  47,  48 
Streptococcus  erysipelatos  (vide 

Streptococcus) 
Streptococcus      pyogenes     (vide 

Streptococcus) 
tetragonus,  51 
Milk,  examination  of,  105 

tubes,  89 
Mycetoma,  46 
Mycoprote'ine,  145 

NENCKI,  144 

Nitrous  acid  iu  cultures,  142 
Nutrient  media :    carbol   gelatine, 
171 

Fermi's  solution,  170 

preparation  of,  83-92 

thymol  gelatine,  169 

OXYGEN,  action  on  bacteria,  5 

PARAFFIN  sections,  64 

actinomycosis,  69,  70 
diphtheritic  membrane,  65,  66 
glanders,  68,  69 
pyaemic  spleen,  70,  71 
tubercle,  64,  65 
embedding,  133,  135 
Peptone  method  (cholera),  101,  102 
Peptones,  150 

tests  for,  150 
Peptone  solution,  preparation  of,  88 

sterilisation  of,  89 
Phagocytes,  72-77 
staining  of,  72.  73 


Phagocytosis,  71,  77 

effect  of  anesthesia  on,  76 
effect  of  heat  on,  74-76 
examination  in  hanging  drop,  71, 

72,  76,  77 
examination  in  stained  specimens, 

72,  73 

Pitfield's  method,  46 
Plates,  agar-agar,  103,  104 
gelatine,  29 
anthrax,  34 
cholera,  42,  44,  104 
quantitative     examination    of 

water,  93-95 
air  and  dust,  109 
Pneumococcus  (vide  Pneumonia) 
Pneumonia,  cultivations,  47 
coccus  of,  50 
fibrin  staining,  51,  52 
tissues,  51,  52 
Pneumonic  sputum,  staining  of,  56 

double  staining  of,  56 
Poisons,   bacterial,  141,   144,  155, 

158,  161,  167,  172 
intracellular,  142 
Potato  tubes,  89 

sterilisation  of,  89 
Preparation  of  nutrient  media,  83-92 

serum  tubes,  155 
Products,  bacterial,  141,  144,  147, 

155,  158,  161,  167,  172 
Proteines,  144-146 
Proteolytic  ferments  and  enzymes, 

168-171 

Proto-albumose  (vide  Albumoses) 
Psorosperms,  124 
Ptomaines,  172-174 
Pus,  staining  of,  19,  20,  48,  49 
with  methylene-blue,  20 
by  Gram's  method,  20 
Putrefaction,  120,  122 
Putrescine,  172-174 

hydrochlorate  of,  174 
Pysemic  abscess,  51 
spleen,  51,  70,  71 
Pyocyanine,  147 
Pyogenic  cocci,  47,  50,  52 
Pyrogallic  acid,  114 


180 


INDEX 


QUININE,  sulphate  of,  160 
ROLL  tubes,  95,  96,  114 

SARCINA  lutea,  6,  8 

staining  of,  10,  11 
Sections,  frozen,  35-37,  51,  57,  62 
(vide  Staining) 

actinomycosis,  45,  69 

anthrax,  35,  37 

celloidin,  45,  46 

diphtheria,  57,  65,  66 

erysipelas,  51 

glanders,  68 

leprosy,  57 

Madura  disease,  46 

micrococcus  tetragonus,  f»l 

mycetoma,  46 

paraffin  (vide  Paraffin),  64 

pneumonia,  51,  52 

pyaemia,  51,  70 

tubercle,  62-65 

typhoid  spleen,  55 
Separation  of  hay  bacillus,  33 
Serum  tubes,  preparation  of,  92 
Shake  cultures,  54,  99 
Silk  threads,  anthrax,  127 
Soil,  examination  of,  115-118 

for     malignant    oedema    bacilli, 
116 

for  tetanus  bacilli,  116 
Spores,  tetanus,  69,  118 

staining  of,  30-32 
Sputum,  pneumonic,  56 

tubercular,  59,  61 
carbolised,  61 
fresh,  59 
watery,  62 
Staining  methods,  Gram's,  17-19,  20 

Van  Ermengem's,  38,  39 

Weigert's  fibrin  staining,  51,  52 

Van  Ketel's,  61,  107 
Staining  of  anthrax  tissues,  27,  28 

celloidin  sections,  35-37 

flagella,  38,  39 

frozen  sections,  35,  37 

hanging  drop  cultures,  17-19 

impression  specimens.  23 


Staining  of  micro  -  organisms  with 

simple  aniline  dyes,  9-]  1 
paraffin  sections,  64-66,  68-71 
psorosperms,  124 
pus,  19,  20 

sputum  (tubercle),  59-62 
Staining  solutions,  aniline  fuchsine. 

30 

aniline  gentian- violet,  18 
carbol  fuchsine,  31 
gentian- violet,  61 
methylene-blue,  68 
Czinzinski's,  35,  36 
Ehiiich's  fuchsine,  30 
eosine,  28,  71,  72 
eosine  and  methylene-blue,  36 
for  flagella  staining,  38,  46 
Gram's  method,  18 
pneumococcus  capsule,  55 
spore  staining,  30,  32 
Weigert's  method,  52 
fuchsine,  9 
gentian -violet,  9 
Loffler's  fuchsine,  13 
methylene-blue,  9 
preparation  of  simple,  9 
Ziehl's  fuchsine,  31 
Staining,  double,   with  rnethylene- 

blue  and  eosine,  28,  35 
with  methylene-blue  and  picro- 

carmine,  36 
Staphylococcus    cereus    flavus,    8, 

47 
Staphylococcus    pyogenes    aureus, 

8,  19,  47,  48 
albus,  47 
Sterilisation  and  putrefaction,  121, 

122 

Sterilisation  by  heat,  141 
by  filtration,  141 
of  beakers,  83 
flasks,  83 
pipette,  93,  94 
test-tubes,  83 

Streptococcus  erysipelatos,  47 
cultivations,  47 
in  hanging  drop,  48 
in  tissues,  51 


INDEX 


181 


Streptococcus     pneumonige     (vide 

Pneumonia) 
pyogenes,  8,  14,  17 
cultures,  47 
Gram's  staining,  17 
hanging  drop,  50 
in  milk,  106 
•     in  sections,  51 
Sugar  filter,  vide  Filter 
Sulphur  dioxide,  disinfectant  action 

of,  129 

Sunlight,  action  on  bacteria,  4 
cholera,  43 
water,  97 

TANK  water  examination,  96 
Tap  water,  examination  of,  93-96 
Temperature,  action  of  bacteria,  3 
Testing  of  filters,  136-138 
Test-tubes,  cleaning  of  new,  81 

used,  82 

sterilisation,  83 
Tetanus  bacillus,  69 

fractional  separation  of,  118 

growth  in  exhausted  flask,  116 

growth  in  hydrogen,  116 

separation  from  soil,  116-118 

staining  of,  69 

spores,  69 

Torula  alba,  6,  8,  13 
Toxiue,  diphtheria,  158,  159 
Trichina  spiralis,  123 
Tryptic  enzymes,  168-171 

ferments,  168-171 
Tubercle,  avian,  67 

bacilli,  67 

frozen  sections,  62,  63 

Gram's  method,  63 

mammalian,  67 

milk,  107 

paraffin  sections,  64,  65 

sputum,  fresh,  59 
carbolised,  61 
watery,  62 

urine,  62 

Van  Ketel's  method,  61,  107 
Typhoid,  action  of  carbolic  acid  on, 
126 


Typhoid  bacilli,  38,  39,  53,  54,  59 
cultivations,  53 
examination  of  water,  98-100 
flagella,  38,  39 
in  milk,  106 
in  spleen,  55,  135 
in  water,  98 
shake  cultures,  54,  99 
staining  of,  54 
25  per  cent  gelatine,  99 

UDRANSKT,  172 

Ulcerative  endocarditis,  51 

Urine,    examination     for    tubercle 

bacilli,  62 
Uschinsky,  158 


VAN  ERMENGEM,  38,  39 

Van  Ketel,  61,  107 

Vibrio     cholerse     Asiatics 

Cholera) 

Fiukler-Prior,  37,  40,  168 
Metchincovi,  37,  40 


(vide 


WATER,  93-104 

bacillus  of  typhoid  fever,  98-100 
bacterium  coli  commune,  98-100 
cholera,  101-104 
effect  of  sunlight  on,  97 
filtration,  136-138 
qualitative  examination,  98-104 
quantitative  examination  of,  93-97 
distilled,  96 
tank,  96 
tap,  93-98 

Weigert's  iodine  solution,  52 
Weigert's    method,   actinomycosis, 

69,  70 

croupous  pneumonia,  51,  52 
diphtheritic  membrane,  66 
fibrin  staining,  51,  52,  66,  69,  70 

YEAST,  161-167 
enzyme.  167,  168 
ferment,  161,  168 
invertine,  161,  168 


Printed  by  R.  &  R.  CLARK,  LIMITED,  Edinburgh. 


MACMILLAN   AND   CO.'S   PUBLICATIONS. 
BY  E.  KLEIN,  M.D. 

THE    BACTERIA    IN    ASIATIC    CHOLERA.     By   E.    KLEIN, 

M.D.,  F.R.S.,  Lecturer  on  General  Anatomy  and  Physiology  in  the  Medical 
School  of  St.  Bartholomew's  Hospital,  Professor  of  Bacteriology  at  the  College  of 
State  Medicine,  London.  Crown  8vo.  55. 

MICRO-ORGANISMS  AND  DISEASE  :  an  introduction  into  the 
study  of  specific  micro-organisms.  With  108  Engravings.  Second  Edition. 
Crown  8vo.  6s. 

THE  ETIOLOGY  AND  PATHOLOGY  OF  GROUSE  DISEASE, 
FOWL  ENTERITIS,  AND  SOME  OTHER  DISEASES  AFFECTING 
BIRDS.  With  60  Illustrations.  8vo.  ys.  net. 


METHODS   OF   PATHOLOGICAL  HISTOLOGY.     By  C.  VON 

KAHLDEN,  Assistant  Professor  in  the  University  of  Freiburg.  Translated  and 
Edited  by  H.  MORLEY  FLETCHER,  M.A.,  M.D.  With  an  Introduction  by  G. 
SIMS  WOODHEAD,  M.D.  8vo.  6s. 

***  A  Companion  Volume  to  Ziegler's  "  Pathological  Anatomy."" 

AERO-THERAPEUTICS  :  OR,  THE  TREATMENT  OF  LUNG 

DISEASE  BY  CLIMATE.  Being  the  Lumleian  Lectures  for  1893,  delivered 
before  the  Royal  Co^ege  of  Physicians.  With  an  Address  on  the  High  Altitudes 
of  Colorado.  By  CHARLES  THEODORE  WILLIAMS,  M.A.,  M.D.,  Oxon.,  F.R.C.P. 
Senior  Physician  to  the  Hospital  for  Consumption  and  Diseases  of  the  Chest, 
Brompton,  late  President  of  the  Royal  Meteorological  Society.  8vo.  6s.  net. 

MATERIALS  FOR  THE  STUDY  OF  VARIATION.  Treated 
with  Especial  Regard  to  Discontinuity  in  the  Origin  of  Species.  By  WILLIAM 
BATESON,  M.A.  Fellow  of  St.  John's  College,  Cambridge.  8vo.  sis.  net. 

PRACTICAL  BOTANY  FOR  BEGINNERS.  By  F.  O.  BOWER, 
D.Sc.,  F.R.S.,  Regius  Professor  of  Botany  in  the  University  of  Glasgow,  Author 
of  "  A  Course  of  Practical  Instruction  in  Botany."  Globe  8vo.  33.  6d. 

THE  STUDY  OF  THE  BIOLOGY  OF  FERNS  BY  THE 
COLLODION  METHOD.  For  Advanced  and  Collegiate  Students.  By 
GEORGE  F.  ATKINSON,  Ph.B.,  Associate  Professor  of  Cryptogamic  Botany  in 
Cornell  University.  8vo.  8s.  6d.  net. 

MACMILLAN  AND  CO.,  LONDON. 


MACMILLAN    AND    CO.'S    PUBLICATIONS. 

A  TEXT -BOOK  OF  PATHOLOGY:  SYSTEMATIC  AND 
PRACTICAL.  By  Professor  D.  J.  HAMILTON.  Copiously  Illustrated.  Vol.11., 
Parts  I.  and  II.  Medium  8vo.  155.  net  each  Part.  (Vol.1,  sis.net.) 

A    TEXT-BOOK    OF    COMPARATIVE    ANATOMY.      By    Dr. 

ARNOLD  LANG,  Professor  of  Zoology  in  the  University  of  Zurich.  With  Preface 
to  the  English  Translation  by  Dr.  ERNST  HAECKEL.  Translated  by  HENRY  M. 
BERNARD,  M.A.,  Cantab.,  and  MATILDA  BERNARD.  Vol.  I.  8vo.  175.  net. 

[Vol.  II.  in  the  Press. 

LESSONS    IN    ELEMENTARY    ANATOMY.     By  ST.    GEORGE 

MIVART,  F.R.S.,  Author  of  "  The  Genesis  of  Species."     Fcap.  8vo.     6s.  6d. 

ELEMENTS  OF  THE  COMPARATIVE  ANATOMY  OF  VERTE- 
BRATES. Adapted  from  the  German  of  ROBERT  WIEDERSHEIM,  Professor  of 
Anatomy,  and  Director  of  the  Institute  of  Human  and  Comparative  Anatomy  in 
the  University  of  Freiburg,  in  Baden.  By  W.  NEWTON  PARKER,  Professor  of 
Biology  in  the  University  College  of  South  Wales  and  Monmouthshire.  With 
additions  by  the  Author  and  Translator.  270  Woodcuts.  Medium  8vo.  125.  6d. 

A  COURSE  OF  INSTRUCTION  IN  ZOOTOMY.  Vertebrata. 
By  T.  JEFFERY  PARKER,  F.R.S.,  Professor  of  Biology  in  the  University  of 
Otago,  N.Z.  With  Illustrations.  Crown  8vo.  8s.  6d. 

AN  INTRODUCTION  TO  THE  OSTEOLOGY  OF  THE 
MAMMALIA.  By  Sir  WILLIAM  HENRY  FLOWER,  F.R.S.,  F.R.C.S.,  Director 
of  the  Natural  History  Department  of  the  British  Museum.  Illustrated.  Third 
Edition.  Revised  with  the  assistance  of  HANS  GADOW,  Ph.D.,  Lecturer  on  the 
Advanced  Morphology  of  Vertebrates  in  the  University  of  Cambridge.  Crown 
8vo.  xos.  6d. 

THE  MYOLOGY  OF  THE  RAVEN  (Corvus  corax  Sinuatus). 
A  Guide  to  the  Study  of  the  Muscular  System  in  Birds.  By  R.  W.  SHUFELDT, 
of  the  Smithsonian  Institute,  Washington,  U.S.A.  With  Illustrations.  8vo. 
135.  net. 

A  COURSE  OF  ELEMENTARY  PRACTICAL  HISTOLOGY. 

By  WILLIAM  FEARNLEY.    Crown  8vo.     75.  6d. 

A  COURSE  OF  ELEMENTARY  PRACTICAL  PHYSIOLOGY 
AND  HISTOLOGY.  By  Prof.  MICHAEL  FOSTER,  M.D.,  F.R.S.,  and  J.  N. 
LANGLEY,  F.R.S.,  Fellow  of  Trinity  College,  Cambridge.  Sixth  Edition. 
Crown  8vo.  75.  6d. 

LESSONS  IN  ELEMENTARY  PHYSIOLOGY.  By  T.  H- 
HUXLEY,  F.R.S.  With  numerous  Illustrations.  Fourth  Edition.  PotSvo.  45.  6d. 

QUESTIONS  ON  HUXLEY'S  LESSONS  IN  ELEMENTARY 
PHYSIOLOGY.  For  the  Use  of  Schools  By  THOMAS  ALCOCK,  M.D.  Fifth 
Edition.  Pot  8vo.  is.  6d. 

MACMILLAN  AND   CO.,  LONDON 


63371 


: 

. 


-  : 


THE  UNIVERSITY  OF  CALIFORNIA  LIBRARY 


